Patent Description:
Proteolysis of proteins is crucial in regulation of cellular processes. Abnormal proteolysis is associated with diseases such as Alzheimer's wherein there is an accumulation of unwanted peptides and/or proteins leading to the formation of aggregates. One of the mechanisms of achieving proteolysis is via the ubiquitin proteolytic system. Proteins and/or peptides are targeted for degradation by adding ubiquitin to the target proteins and/or peptide which removes unwanted proteins.

Chimeric constructs have been developed as a novel way of specifically targeting proteins and/or peptides for proteolysis. PROTACs (proteolysis targeting chimera) are chimeric constructs having a target protein binding moiety and an E3 (ubiquitin) ligase binding moiety, bringing the target protein in close proximity to the E3 ligase for ubiquitination (<CIT>). As E3 ubiquitin ligases play a crucial role in the induction of degradation and therefore regulation of cellular processes, they are an attractive therapeutic target.

There are approximately <NUM>-<NUM> human E3 ligases as estimated by human genome sequence analysis. These can be divided into four families: ECT-domain E3s (HECT E3s; homologous to E6AP C terminus E3s), U-box E3s, monomeric RING E3s (RING-finger E3s) and multi-subunit E3s (<CIT>). Currently only a very limited number of known E3 ligases have been demonstrated to be linkable in a targeted degradation approach (e.g. Mouse double minute <NUM> homolog (MDM2), Von Hippel-Lindau (VHL), Cereblon (CRBN), F-box/WD repeat-containing protein 1A (FBXW1A; beta-TrCP1) and Cellular Inhibitor of Apoptosis Protein <NUM> (c-IAP1; BIRC2)) (see <CIT>, <CIT>, <CIT> and <CIT>; <NPL>; <NPL>). MDM2, VHL, CRBN, FBXW1A and c-IAP1 ligases all belong to the RING E3 ligase family, the largest family of E3s (<NPL>). Despite the crucial role played by E3 ligases in cellular regulation, to date the majority of PROTACs are designed to recruit either VHL or CRBN to induce ubiquitination (<NPL>). Whilst the basic PROTAC technology has been a promising advance in drug discovery, this use of a restricted subset of E3 ligases, belonging to only one family, limits the utility of the technology. The majority of known E3 ligases are yet to be exploited, meaning that none from the HECT, U-box or multi-subunit E3 family have been recruited via PROTACs. Further, it is expected that currently unknown or uncharacterised E3 ligases will show variation in degradation efficiency and selectivity for target proteins over those previously studied, potentially leading to improvements in degradation and therapeutic efficacies (<NPL>).

There is a need therefore to develop methods to identify new E3 binding moieties, and/or the E3 ligases with which they interact, in order to exploit the full potential of E3 ligases in a PROTAC-based targeted degradation approach. Such methods may further permit the identification and exploitation of currently unknown or uncharacterised E3 ligases. In addition, the identification of new E3 binding moieties may enable differential expression among E3 ligases to be exploited, in order to target therapeutic PROTACs to a specific tissue type, cell type or developmental stage.

It is against this background that the present inventors have developed a new functional screening method for candidate E3-binding moieties.

Related art also includes the following papers, but none of these disclose the present invention as claimed in the present specification.

Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation.

Identification of a Sequence Element from p53 That Signals for Mdm2-Targeted Degradation.

Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

The invention is defined in the claims. Hereinafter the term "embodiment" only relates to an embodiment of the invention if it falls in the scope of the claims. Otherwise it relates to a mere embodiment of the disclosure.

In a first aspect, the invention provides a method for determining if a peptide binds or is capable of binding to a ubiquitin protein ligase (E3) and thereby leads to degradation of a test protein, wherein the peptide is between about <NUM> and <NUM> amino acids in length,
the method comprising:.

In one embodiment of the method of the first aspect of the invention, the hybrid polypeptide comprises a domain being the candidate peptide, a domain being the test protein and a domain being the peptide linker, thereby providing the candidate peptide functionally linked to the test protein.

In another embodiment of the method of the first aspect of the invention, the hybrid polypeptide comprises a domain being the candidate peptide, a domain being a test protein-binding (poly)peptide, and a domain being a peptide linker, and wherein, when the hybrid polypeptide and the test protein are present in the cell, the hybrid polypeptide binds to the test protein via the test protein-binding (poly)peptide, thereby providing the candidate peptide functionally linked to the test protein.

The amino acid sequence of the E3-binding peptide may not consist of an amino acid sequence of a known E3-binding peptide; thereby a new E3-binding peptide may be identified by the methods of the invention.

Functional linkage/functionally linked as used herein refers to peptides, polypeptides or proteins that interact with each other, either due to being physically linked (e.g. via a linker peptide, or due to being separate domains of a fusion protein), or due to their coming into proximity (e.g. in vitro or intracellularly) and thereby interacting and/or forming a complex. Where peptide, polypeptides or proteins form parts of a (recombinant) fusion protein, this may be generated by genetic fusion. This genetic fusion may be at any appropriate point in the sequence, not simply limited to the termini. Those skilled in the art will appreciate that the fusion protein may be expressed recombinantly in an appropriate cell. "Recombinant" as used herein to describe a protein or peptide means one expressed from a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (<NUM>) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (<NUM>) is linked to a polynucleotide other than that to which it is linked in nature.

In accordance with any aspect of the invention, providing in a eukaryotic cell a candidate peptide functionally linked to a test protein may comprise providing one or more nucleic acid encoding such a candidate peptide and test protein.

In one embodiment of a method described herein, the method further comprises identifying one or more E3 to which the E3-binding peptide binds or is capable of binding.

In one embodiment, the method may further comprise identifying the E3-binding peptide. Suitable identification methods may include sequencing for example by next-generation sequencing (NGS) or Sanger sequencing, by PCR, or by mass spectrometry etc. Where the candidate peptide is provided as one of a plurality of candidate peptides, each spatially separated and presented in a defined position (e.g. within an array) for example in a microtiter plate or on a chip, the E3-binding peptide may be identified by reference to its position within the array, following detection of the amount of test protein in the cell (detection being carried out, for example, by microscopy, flow cytometry, elisa or Western blot).

Suitably, the peptide that is capable of binding to a ubiquitin protein ligase (E3) is thereby capable of leading to or recruiting ubiquitination of a test or reporter protein.

In one embodiment of any aspect of the invention, the candidate peptide is between about <NUM> and <NUM> amino acids in length, preferably wherein the candidate peptide is about <NUM> to <NUM> amino acids in length. In another embodiment the candidate peptide is about <NUM> to <NUM> amino acids in length. Suitable candidate peptides for use in accordance with the invention will be known to those skilled in the art.

Suitably the "test" protein or peptide is a target protein. Such a target protein may, for example, be labelled with an antibody, a tag, or may be a fluorescent protein fusion. In other embodiments, the test protein may be a reporter protein. In yet other embodiments, the test protein may be a target-reporter fusion protein. In some embodiments the test protein may comprise a plurality of lysine residues within its native amino acid sequence. In all aspects of the invention the test protein is genetically engineered to comprise a plurality of lysine residues. Such lysine residues may be present at the N-terminus or the C-terminus of the test protein, or internally within the test protein amino acid sequence.

As referred to herein, "protein", "(poly)peptide" and "peptide" may be used interchangeably.

Suitably the eukaryotic cell is provided with a candidate peptide under conditions enabling ubiquitination of proteins by an E3. In some embodiments, an E3 is the E3 identified in accordance with a method described herein.

Suitably, in the detection step, the amount of the test, e.g. target or reporter, protein present is measured and compared to a control.

Suitably, a candidate peptide that binds or is capable of binding to an E3 is capable of leading to and/or recruiting ubiquitination of the test, e.g. reporter, protein. Thus the 'candidate peptide' is identified as an E3-binding peptide.

In some embodiments of any aspect of the invention, the amino acid sequence of the E3-binding peptide is not comprised in the amino acid sequences of a known substrate of the E3, such as a native substrate.

In one embodiment, a method in accordance with any aspect of the invention comprises or further comprises identifying the E3-binding peptide (degron). Degron (peptide) as used herein refers to a candidate peptide containing an E3 binding sequence (i.e. an E3-binding peptide) and having the potential to recruit an E3 ligase. When a degron peptide / E3-binding peptide is linked to either (i) a test protein binding moiety e.g. a test protein binding (poly)peptide (thereby forming a PROTAC), or (ii) a test protein itself, the test protein and the E3 ligase are brought into close proximity.

In one embodiment, a method described herein further comprises using the E3-binding peptide in a screening method against a target protein, for example a therapeutic target protein. Such a screening method may comprise: fusing the E3-binding peptide to each of a plurality of candidate target protein binding peptides in order to produce a peptide-peptide fusion library. This peptide-peptide fusion library may then be screened for the ability to degrade the target protein when introduced into eukaryotic cells under conditions enabling ubiquitination of proteins by an E3. By this method therefore, E3-binding peptides, and target protein binding peptides, may be determined. An E3-binding peptide - target protein binding peptide fusion found to lead to degradation of the target protein in the screen may be used or developed further, for example as a composition for use in treatment of diseases related to the target protein.

In another embodiment, a method described herein further comprises identifying an E3 to which the E3-binding peptide binds or is capable of binding.

Suitable methods for identifying an E3 will be familiar to those skilled in the art and may include, for example, using techniques such as pull-down, Y2H, Y3-hybrid, or through bioinformatics approaches using databases such IntAct, for example. Identification can be confirmed using a candidate approach with knockdown of a target using shRNA, for example, to confirm that this restores test protein levels.

In another embodiment of any aspect of the invention, the test protein is a reporter protein, a reporter protein fusion, or is functionally linked to a reporter protein, and wherein detecting the amount of test protein present in the cell comprises detecting the amount of reporter protein present in the cell.

In one embodiment, a method in accordance with the invention further comprises selecting a cell based on a reduction in the amount of test protein present in the cell. In one embodiment, a method in accordance with the invention further comprises selecting a cell based on a reduction in the amount of reporter protein present in the cell, wherein the test protein is a reporter protein, a reporter protein fusion, or is functionally linked to a reporter protein.

In some embodiments, the reduction in the amount of test or reporter protein may be analysed in the absence and presence of a proteasomal inhibitor such as MG132, as described herein, for example, so as to ensure that a low test protein or low reporter signal is caused by degradation of the protein via the UBP system and not by other means (such as low expression, autophagy etc.). Other proteasomal inhibitors will be known to those skilled in the art, and suitably include Bortezomib, Z-LLF-CHO, Lactacystin and inhibitors of specific <NUM> proteasomal proteolytic functions such as Chymotrypsin-like, Trypsin-like and Caspase-like inhibitors. The terms 'proteasomal inhibitor' and 'proteasome inhibitor' are used interchangeably herein.

In some embodiments, cells for analysis may be separated using fluorescence-activated cell sorting (FACS), or magnetic cell sorting, or may be analysed using a cell proliferation or survival assay.

Suitably, the eukaryotic cell for use in accordance with the method of any aspect or embodiment is a mammalian cell, suitably a human-derived cell.

In some embodiments the test protein comprises a suitable reporter moiety, including, for example, a fluorescent or luminescent protein, or a protein that can be stained with an antibody to be used in a fluorescent or colorimetric readout, or with an affinity tag, a suicide gene, an antibiotic resistance marker or an enzyme.

Hybrid polypeptide as used herein refers to a polypeptide containing domains from more than one source. Suitably, the hybrid polypeptide includes a domain being the candidate peptide, a domain being the test protein and a domain being a peptide linker, thereby providing the candidate peptide functionally linked to the test protein. The hybrid polypeptide may include a domain being a test protein that is linked, suitably through a covalent interaction (for example as a fusion protein generated by genetic fusion), to a domain being the candidate peptide, via a peptide linker. In some embodiments, the linker has a length of between about <NUM> and <NUM> amino acids. In some embodiments, at least <NUM>, <NUM>, <NUM>, <NUM> or <NUM> amino acids of the test protein may be lysine residues.

Flexible linkers may be used when the linked domains require movement. They usually consist of small non-polar (e.g.: Gly) or polar (eg: Ser, Thr) amino acids, where the small size provides flexibility (<NPL>). Any suitable flexible linker may be used, with the nature and length appropriate to the entities concerned. In some cases rigid linkers may be preferred, as they can assist with providing protein separation. Rigid linkers have a secondary structure. One of the most common rigid linkers is (EAAAK)n (where n is the number of repeats) which adopts an α-helical structure (<NPL>). Other rigid linkers may include proline rich sequences such as (XP)n, where X is any amino acid but preferentially Ala (A), Lys (K) or Glu (E), where the proline provides conformational constraint (Chen at al.

In other embodiments, the hybrid polypeptide includes a domain being the candidate peptide, a domain being a test protein binding (poly)peptide and, a domain being a peptide linker, wherein, when the hybrid polypeptide and the test protein are present in the cell, the hybrid polypeptide binds to the test protein via the test protein binding (poly)peptide; thereby providing the candidate peptide functionally linked to the test protein.

In another embodiment, the test protein comprises a protein associated with a disease, disorder or condition when expressed or over-expressed in a eukaryotic cell. Suitably, the protein of interest is one selected from a class of proteins selected from the group consisting of: members of an oncogenic pathway; viral host factors; viral proteins; mis-folded proteins; aggregating proteins; toxic proteins; proteins involved in immune recognition, immune response or auto-immunity; shuttle proteins.

The candidate peptide, the test protein, the test protein binding (poly)peptide and/or the hybrid polypeptide in accordance with any aspect of the invention may be provided by expressing in the eukaryotic cell one or more nucleic acid molecule encoding the candidate peptide, the test protein, the test protein binding (poly)peptide and/or the hybrid polypeptide. Suitably, the nucleic acid sequences encoding the candidate peptide, the test protein, the test protein binding (poly)peptide and/or the hybrid polypeptide may be encompassed in different plasmids/vectors such that transfection of a eukaryotic cell with each plasmid/vector will result in each component (peptide/polypeptide/protein) being produced by the cell, when it is cultured in suitable conditions. In other embodiments, a single plasmid/vector may comprise a combination of one or more nucleic acid sequence encoding the candidate peptide, the test protein, the test protein binding (poly)peptide and/or the hybrid polypeptide, such that transfection of a eukaryotic cell with this plasmid/vector will result in each component being produced by the cell, when it is cultured in suitable conditions. In other embodiments, an entire fusion protein coding sequence comprising: (i) the candidate peptide coding sequence, and (ii) the test protein coding sequence or the test protein binding (poly)peptide coding sequence, suitably joined by the optional peptide linker coding sequence, may be provided in one plasmid/vector, such that transfection of a eukaryotic cell with this plasmid/vector will result in the entire fusion protein being produced by the cell, when it is cultured in suitable conditions.

In another aspect, the invention provides a library of nucleic acid molecules as defined in the claims for use in a method in accordance with the invention. Suitable nucleic acid molecules may comprise a construct encoding the candidate peptide (functionally) linked to the test protein or the test protein binding (poly)peptide. Where the candidate peptide is one of a plurality of candidate peptides, for example a library of candidate peptides, a plurality of nucleic acid molecules may be provided, each comprising the coding sequence for one candidate peptide. Described herein is a vector comprising such a nucleic acid.

Suitably, the E3-binding peptide is identified from the nucleotide sequence of the nucleic acid molecule encoding the (relevant) candidate peptide. Suitable methods of identifying the nucleotide sequence will be known to those skilled in the art and include next-generation sequencing (NGS), Sanger sequencing, microarray and similar hybridisation-based approaches.

In a method in accordance with any aspect or embodiment of the invention, additionally a reduced amount of the test protein is determined by comparison to a control, to a threshold and/or to a reference distribution. Suitably, a reduced amount of test protein is determined by comparison to a control. In one embodiment, the test protein comprises a first reporter protein. Suitably, where the test protein comprises a first reporter protein, the control may comprise a control eukaryotic cell comprising the test protein comprising the first reporter protein but not functionally linked to the candidate peptide. Alternatively, where the test protein comprises a first reporter protein the control may comprise a second reporter protein present in the eukaryotic cell that is not functionally linked to the candidate peptide. In some embodiments, the first reporter protein and the second reporter protein are fluorescent proteins having different excitation and/or emission frequencies. Suitable reporter proteins will be known to those skilled in the art and include but are not limited to GFP, EGFP, RFP, DsRed, mCherry etc..

In another embodiment, the control may comprise a control eukaryotic cell comprising the candidate peptide functionally linked to the test protein, but under conditions that prevent ubiquitination of proteins by an E3, preferably wherein the conditions comprise treating the control eukaryotic cell with a proteasome inhibitor, e.g. MG132.

In a further embodiment of any aspect of the invention, the candidate peptide functionally linked to the test protein is provided as a member of a library of candidate peptides as defined in the claims, suitably each functionally linked to the test protein. The candidate peptides comprised in the library have a length of between about <NUM> and <NUM> amino acids, preferably between about <NUM> and <NUM> amino acids, and have an amino acid sequence being a region of a sequence selected from the amino acid sequence of a naturally occurring protein of one or more organisms; wherein the library comprises nucleic acids that encode for a plurality of at least <NUM>,<NUM> different such peptides, and wherein the amino acid sequence of each of at least <NUM> of such peptides is a sequence region of the amino acid sequence of a different protein of a plurality of different such naturally occurring proteins. Some candidate peptides comprised in the library may be derived from one or more starting peptides from which systematic variants have been generated. The candidate peptides may be derived from proteins known to bind to E3 ligases. The library may comprise some random amino acid sequences.

Suitably each candidate peptide functionally linked to the reporter protein, is provided by a library of nucleic acids capable of expression in the eukaryotic cell. In some embodiments, each nucleic acid of the library encodes for a hybrid polypeptide; wherein the hybrid polypeptide includes a domain being a candidate peptide, a domain being the test protein and, a domain being the linker. In some embodiments, the hybrid polypeptide includes a domain being the candidate peptide, a domain being the reporter protein binding (poly)peptide and, a domain being the linker.

In another aspect of the invention, there is provided a library of (e.g. synthetic) nucleic acids, each nucleic acid comprising a coding region of defined nucleic acid sequence encoding for a peptide being a hybrid polypeptide comprising: (i) a candidate peptide being a member of a library of candidate peptides; (ii) a test protein or a test protein binding (poly)peptide; and (iii) a peptide linker, in each case independently as recited in any one of claims <NUM> to <NUM>; wherein the library of candidate peptides comprises peptides having a length of between about <NUM> and <NUM> amino acids, and have an amino acid sequence being a region of a sequence selected from the amino acid sequence of a naturally occurring protein of one or more organisms; wherein the library comprises a plurality of at least <NUM>,<NUM> different such peptides, and wherein the amino acid sequence of each of at least <NUM> of such peptides is a sequence region of the amino acid sequence of a different protein of a plurality of different such naturally occurring proteins;
and wherein the amino acid sequence of the test protein is genetically engineered to comprise a plurality of lysine residues, wherein the test protein is genetically engineered to comprise an N-terminal lysine-rich region derived from human nitric oxide synthase (NOS1).

In another aspect, the invention provides a library of peptides, wherein the peptides are the hybrid polypeptides encoded by the library of nucleic acids in accordance with the invention.

Suitably in a library in accordance with any aspect or embodiment of the invention, the individual members thereof are in a pooled format. For example, a "pooled format" (or "pooled form") includes those where the individual members (or subset of members) thereof are in admixture with other members (or subsets); for example, a solution (or dried precipitate thereof) of such members contained in a single vessel, or a population of host cells containing recombinant vectors according to the present invention.

In another embodiment, the individual members of a library in accordance with the invention are spatially separated. A "spatially separated" library can be considered as a library in which a plurality of members (or sub-sets of members) of the library are physically separated, suitably in an ordered manner, from each other. Examples of a spatially separated library include those where individual members (or sub-sets of members) are comprised in individual wells of one or more mictrotitre plates, are arrayed on a solid surface or are bound (in an ordered manner) to a silicon wafer. In another embodiment, individual members (or sub-sets of members) in a library in accordance with the invention are each individually addressable; that is they can be retrieved (e.g. without undue searching or screening) from the library. Suitable methods for addressing or interrogating the library in accordance with the invention may include Next Generation Sequencing (NGS), PCR etc. Also, when the library is present in a spatially separated format (or form), the individual members (or sub-sets of members) may be "individually addressable" by knowing the spatial location of the applicable individual member (or sub-set). In either of these embodiments, use of a computer program, data file or database (such as those utilising a computer-readable medium or data-processing system, of the invention) can facilitate the retrieval of individual members (or sub-sets of members) that are comprised in an individually addressable library of the invention.

In another aspect, there is provided a population of cells, transformed with the library of nucleic acids in accordance with the invention, or expressing the library of peptides in accordance with the invention. Suitable cells include eukaryotic cells, such as mammalian cells, human cells e.g. HEK293A cells, immortalized cells and primary cells (ie fibroblasts, glial cells, neuronal cells). Suitable cells for use in accordance with the invention will be known by those skilled in the art. In one embodiment, the host cells will be suitable for transient expression. In another embodiment, host cells will be those cells which are capable of forming stable cell lines.

Described herein is a container or carrier comprising a library in accordance with any aspect or embodiment of the invention or a population of cells in accordance with any aspect or embodiment of the invention. Suitable containers include a microtiter plate or a silicon carrier.

Described herein is a computer-readable medium having information stored thereon comprising: (i) the nucleic acid sequences comprised in the library in accordance with any aspect or embodiment of the invention and/or (ii) the amino acid sequences comprised in the library of peptides in accordance with any aspect or embodiment of the invention.

Described herein is a data-processing system for storing and/or processing information comprising: (i) the nucleic acid sequences comprised in the library of nucleic acids in accordance with any aspect or embodiment of the invention and/or (ii) the amino acid sequences comprised in the library of peptides in accordance with any aspect or embodiment of the invention.

Suitably the computer-readable medium, or the data-processing system described herein further comprises information identifying the E3 or E3s that each peptide being an E3-binding peptide is capable of binding.

In some embodiments, a reduction in the amount of test protein present in the cell is analysed in the presence of a proteasome inhibitor (e.g. MG132).

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the following Figures and Examples.

E3 ubiquitin ligases (E3s) regulate cellular homeostasis, including cell cycle regulation, cell survival, cell differentiation, DNA repair pathways and innate and acquired immunity. In disease, such as cancer, a number of these proteins such as MDM2, BRCA1, and Von Hippel-Lindau tumor suppressor are dysregulated.

Known E3 ubiquitin ligases include E3A, mdm2, Anaphase-promoting complex (APC), UBR5 (EDD1), SOCS/ BC-box/ eloBC/ CUL5/ RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, Parkin.

A degron is a portion of a protein that is used by E3 ubiquitin ligases to target said protein for degradation. Known degrons include short amino acid sequences, structural motifs and exposed amino acids (often lysine or arginine) located anywhere within the protein. Posttranslational modifications, including phosphorylation, represent the most common mechanism for control of substrate recognition by E3 ligases. Other forms of posttranslational modification include acetylation, sumoylation or glycosylation.

The E3 ubiquitin ligase recruits ubiquitin conjugating, or E2, enzymes and then catalyzes the transfer of ubiquitin molecules from E2 onto lysine residues of the target protein, or, to a preceding ubiquitin moiety on the target protein. This results in the formation of poly-ubiquitin chains, or multi- or mono-ubiquitylation events, each of which have a different functional outcome. Poly-ubiquitination targets substrates for destruction by the proteasome.

PROTACs are hetero-bifunctional compounds with bivalent selectivity. They consist of:.

The binding of PROTACs to their E3 ubiquitin ligase and target protein substrates brings the E3 ubiquitin ligase and target protein into close proximity to one another as a ternary complex, thus facilitating E3-mediated ubiquitination of the target protein. As the two ligands do not interact, the linker determines the relative orientation of the two proteins in the ternary complex. In most cases, the E3 ligase has not evolved to bind the protein substrate, therefore the interaction critically depends on the specificity of the bridging PROTAC to both the E3 ubiquitin ligase and the protein substrate. The PROTAC essentially acts as a catalyst for the degradation of the target protein as it is able to dissociate from the binding proteins after ubiquitination of the target protein and is able to repeat its action on the next target protein.

The term PROTAC was first coined by Crews and his colleagues in <NUM>, where their synthesised compound PROTAC-<NUM> contained a SCFβ-TRCP binding phosphopeptide and small-molecule ovalicin that covalently bound MetAP-<NUM>. Degradation of MetAP-<NUM> using Xenopus egg cell lysate was dependent upon PROTAC-<NUM> (<NPL>).

As cell permeability is an essential criterion for use therapeutically, cell permeable PROTACs were later developed. These exist as small peptides or small molecules, both of which are sufficiently small enough to pass through the cell membrane. These are often based on known interactors with E3 ubiquitin ligases such as Hypoxia-inducible factor <NUM>. Once peptide ligands (moieties) to the E3 ubiquitin ligase and ligands (moieties) to the target protein are identified, the PROTAC is generated by the expression of the moieties conjugated together through use of a linker. Alternatively, the moieties may be replaced by small molecule mimics.

The current challenges and limitations of PROTACs include their high molecular weight and polar surface areas, which are associated with poor cell permeability, bioavailability and tissue distribution. Furthermore, the published PROTAC molecules all rely on the utilisation of a small number of ubiquitin E3 ligases, each with different degradation efficiencies. As there are an estimated <NUM>-<NUM> human ubiquitin E3 ligases, it is likely that as yet, unidentified ubiquitin E3 ligases could exist with degradation efficacies vastly superior to those in current use.

Furthermore, the interaction of a particular E3 ligase with a particular polypeptide or protein does not guarantee proteasomal degradation of that substrate. Therefore combining a degron known to recruit a particular E3 with a peptide known to bind a target of interest does not always result in a functional PROTAC. The development of a high-throughput functional screen capable of identifying a large repertoire of degrons, that recruit one or more E3 ubiquitin ligases thereby accessing a greatly increased pool of usable E3s, is of great interest.

An endogenously expressed PROTAC to specifically degrade Tankyrase <NUM> (TNKS2) was developed. A plasmid encoding for a peptide composed of a target (TNKS2) binding moiety, a linker, and an E3 ligase binding moiety was expressed in HEK293A cells. The target binding moiety peptide was chosen by searching the Protein Data Bank for protein-peptide structures. A short peptide corresponding to residues <NUM> - <NUM> of human SH3 domain-binding protein <NUM> (3BP2)(LPHLQRSPPDGQSFRS) as set out in SEQ ID NO: <NUM>, is known to bind to TNKS2 (residues <NUM> - <NUM>), with <NUM> affinity in fluorescence polarization binding assays (Guettler et al. This was selected as the target binding moiety (peptide or ligand). A peptide derived from HIF1α (ALAPYIP) as set out in SEQ ID NO: <NUM>, which binds to Von Hippel-Lindau (VHL), the substrate recognition portion of a Cullin-RING (CLR) E3 ligase, was used as the E3 ligase binding moiety. <FIG> illustrates the overview of the proof of concept experiment.

A DNA construct encoding for the TNKS2 binding sequence (3BP2<NUM>-<NUM>) at the N-terminus as set out in SEQ ID NO: <NUM>, a GGGGSS linker as set out in SEQ ID NO: <NUM>, and the E3 binding sequence (ALAPYIP) as set out in SEQ ID NO: <NUM>, at the C-terminus was cloned into pMOST25 vector (the full PROTAC as set out in SEQ ID NO: <NUM>). pMOST25 is a a lentiviral vector based on pCDH EF1a (System Biosciences) where the promoter was swapped for CMV and the MCS replaced by a new cassette with flanking NGS primer sites. Control constructs which (i) lacked the ALAPYIP E3 binding sequence (3BP2<NUM>-<NUM>) as set out in SEQ ID NO: <NUM> or (ii) contained a double mutation which is known to disrupt HIF1α - VHL interaction (3BP2<NUM>-<NUM> - ALAAAIP) as set out in SEQ ID NO: <NUM> were similarly cloned (Table <NUM>). Neither control construct can bind to the E3 ligase and therefore there should be no change in the level of TNKS2 when cells are transduced with these. HEK293A cells were collected <NUM> hours after transfection, and levels of TNKS2 target protein were assessed by Western Blot (<FIG>). Membranes were probed with primary antibodies against TNKS2 (Abcam ab155545), or actin (Abcam ab179467) as a loading control. Expression of the DNA encoding the full PROTAC (comprising the TNKS2 binding sequence, the linker and the E3 ligase binding sequence (3BP2<NUM>-<NUM> - ALAPYIP)) lead to markedly decreased levels of TNKS2, compared to cells transfected with no DNA or either of the two control constructs 3BP2<NUM>-<NUM> and 3BP2<NUM>-<NUM> - ALAAAIP. This demonstrates that expression of a DNA encoding for a peptide with a target binding sequence, a linker, and an E3 ligase binding sequence (e.g. 3BP2<NUM>-<NUM> - ALAPYIP) can efficiently and rapidly degrade the target protein in cells.

HEK293A cells were transfected with either (i) no DNA, or plasmids encoding for (ii) 3BP2<NUM>-<NUM> (LPHLQRSPPDGQSFRS; the TNKS2 binding sequence) plus linker as set out in SEQ ID NO: <NUM>, or (iii) either 3BP2<NUM>-<NUM> linked to LDPETGEYL (the TNKS2 binding sequence plus a Kelch-like ECH-associated protein <NUM> (Keap1) binding sequence)as set out in SEQ ID NO: <NUM> or 3BP2<NUM>-<NUM> linked to ALAPYIP (the TNKS2 binding sequence plus a VHL binding sequence) as set out in SEQ ID NO:<NUM>. Keap1 and VHL are depicted by E3 ligase A or B, respectively, in <FIG>.

<NUM> hours after transfection, cells were harvested and analyzed by Western Blotting. Membranes were probed with primary antibodies against TNKS2 (Abeam ab155545), or actin (Abeam ab179467) as a loading control. After the addition of HRP-conjugated secondary antibodies, protein signals were detected by chemiluminescence. TNKS2 levels were unaffected by expression of 3BP2<NUM>-<NUM>-LDPETGEYL (SEQ ID NO: <NUM>) indicating that Keap1 (i.e. E3 ligase A) is unable to lead to TNKS2 target degradation (<FIG>). However, 3BP2<NUM>-<NUM>-ALAPYIP peptide led to TNKS2 degradation (<FIG>). Thus, VHL (i.e. E3 ligase B) can efficiently functionally degrade TNKS2 target. This demonstrates that E3 ligases have substrate selectivity and not all E3 ligases can be exploited to degrade any given target. Indeed, there is a need to uncover many moieties which bind to a variety of E3 ligases in order to fully exploit the therapeutic potential of PROTACs to degrade many diverse targets.

A screen was developed to look for peptides (degrons) which can bind to E3 ligases and therefore initiate degradation of a GFP reporter. <FIG> illustrates the E3 reporter library polypeptides, expressed from the E3 reporter library constructs, each polypeptide comprising: an N-terminal Lysine-rich region (depicted as (K)x), GFP (constant region), and a variable region comprising a candidate peptide derived from the "DegroPEx" library at the C-terminus.

The Lysine-rich region was derived from human nitric oxide synthase (NOS1) and serves as a target for ubiquitination by E3 ligases as it contains a large number of Lysine residues. The two DegroPEx libraries each consist of <NUM>,<NUM> constructs encoding peptides derived from proteins known to bind to E3 ligases, one library consisting of peptides <NUM> amino acids in length, the other consisting of peptides <NUM> amino acids in length. To screen for candidate peptides (degrons) that can functionally recruit E3 ligases, cells infected with the E3 reporter-library constructs were sorted by fluorescence-activated cell sorting (FACS) according to their GFP fluorescence levels. Cells expressing E3 reporter library polypeptides comprising functional degron peptides recruited an E3 ligase, which in turn polyubiquitinated the Lysine-rich region of the E3 reporter polypeptide and directed the whole E3 reporter polypeptide to the proteasome (<FIG>), resulting in decreased GFP fluorescence levels compared to cells expressing E3 reporter library polypeptides with candidate peptides that did not function as E3-binding peptides.

The E3 reporter library construct was derived from a pMOST25-Lysine-rich region-emGFP vector. The configuration used in the screen was:
<NUM>'-KOZAC-ATG- Lys-region-Xbal-emGFP-EcoRl-ePCR/NGS fwd primers-BamHl--candidate peptide library sequence-STOP-<NUM>'.

The region underlined (ePCR/NGS fwd primers) is where the primers bind for the sequencing of the candidate peptide sequence. This also acts as a "linker" between the GFP and the peptide library.

When transcribed, these constructs result in the E3 reporter library polypeptides as represented in <FIG> from C-terminus to N-terminus orientation, and having the following amino acid sequence (SEQ ID NO <NUM>):
<IMG>.

The C-terminal SAAT amino acids (underlined) were not present in constructs including a candidate peptide as these were deleted following BamHI digestion.

The Lysine-rich region sequence corresponds to human NOS1 residues <NUM>-<NUM> (R354A/G357D/R376K mutant). The "DegroPEx" candidate peptide libraries were separately cloned into the construct in order to be expressed at the C-terminal end of the E3 reporter library polypeptides. Lentivirus was produced and HEK293A cells were infected at an MOI of <NUM> with a library-fold coverage of at least <NUM>. Once antibiotic selection was completed, cells were either treated with DMSO or treated with <NUM> MG132, a proteasomal inhibitor, for <NUM> hours. Cells were then harvested and sorted by FACS into low and high GFP populations, separately for DMSO- and MG132-treated populations. The genomic DNA was extracted from the populations of interest and analyzed by Next Generation Sequencing (NGS). E3 reporter library polypeptides comprising candidate peptides that can functionally recruit E3 ligases (degrons) are expected to be <NUM>) enriched in the low GFP population in DMSO-treated cells and <NUM>) shift to the high GFP population upon MG132 treatment. <NUM> hit peptides were identified from screening the libraries (<FIG> and <FIG>).

To further demonstrate the feasibility of the screen described in Example <NUM>, a nucleotide sequence encoding a peptide known to bind to an E3 ligase was cloned into the BamHl site of the E3 reporter library construct described in Example <NUM> in lieu of the "DegroPEx" candidate library, in order to generate the E3 reporter LDLEMLAPYIP construct. The LDLEMLAPYIP degron peptide (SEQ ID NO: <NUM>) corresponds to the HIF1α Von Hippel-Lindau (VHL) binding sequence. VHL is the substrate binding domain of a Cullin-RING (CLR) E3 ligase. HEK293A cells were infected with the E3 reporter-LDLEMLAPYIP construct using lentivirus, leading to expression of the E3 reporter LDLEMLAPYIP polypeptide, which then bound to VHL CLR E3 ligase and resulted in the polyubiqutination and degradation of the polypeptide, illustrated by markedly lower GFP fluorescence levels compared to reporter-only (GFP alone, 'E3 reporter') cells (<FIG>). This demonstrates that tagging the reporter with a known E3 binding peptide (degron) successfully results in proteasomal degradation of the reporter, in an E3 dependent manner, and can be directly assessed by monitoring GFP levels.

GFP was used as the target (test protein) in this study. Plasmids encoding GFP alone ('no peptide control' samples), or GFP tagged with either a negative control candidate peptide (a candidate peptide from the <NUM>-mer peptide library that did not lead to degradation of the test protein in a previous screen) or a validated 'hit' candidate peptide ('Hit (#<NUM>)', also from the <NUM>-mer peptide library; amino acid sequence MQNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMA as set out in SEQ ID NO: <NUM>) were transduced into HEK293A cells using lentivirus. The tagged GFP constructs were similar to the E3 reporter library polypeptides generated in Example <NUM> above, comprising the candidate peptides cloned C-terminally to GFP after NGS primer binding sites which also serve as a linker.

GFP-peptide expressing cells were either untreated (DMSO treatment only; 'Peptide + vehicle') or treated with the proteasome inhibitor MG132 (<NUM> in DMSO, for <NUM>) (`Peptide + MG132'), and were then analyzed by FACS for GFP levels (<FIG>). Tagging of GFP with a negative control candidate peptide had no effect on GFP levels compared to 'no peptide control' cells, in any condition (<FIG>). However, addition of a validated hit candidate peptide (Hit (#<NUM>)) to GFP caused marked degradation of GFP (`Peptide + vehicle') compared to 'no peptide control' cells in untreated samples; addition of MG132 to cells expressing GFP-Hit (#<NUM>) restored GFP levels back to that of the 'no peptide control' cells (<FIG>). These results demonstrate that Hit (#<NUM>) is an E3-binding peptide and directs target degradation via the Ubiquitin Proteasome System.

Plasmids encoding for GFP alone (control), or GFP tagged with one of the validated candidate peptide hits from the screening method, Hit (#<NUM>) (see Example <NUM>) or Hit (#<NUM>), were transduced into HEK293A cells using lentivirus. Cells expressing tagged GFP were either untreated (DMSO treatment only) or treated with the proteasome inhibitor MG132 for <NUM> hours, after which they were harvested and analyzed by Western Blotting. Membranes were probed with primary antibodies against GFP (CST #<NUM>), or actin (Abcam ab179467) as a loading control. After the addition of HRP-conjugated secondary antibodies, protein signals were detected by chemiluminescence (<FIG>). Expression ofGFP-Hit (#<NUM>) or GFP-Hit (#<NUM>) led to almost complete depletion of GFP in the absence of MG132 compared to GFP (control) expressing cells. GFP levels were restored in both tagged GFP cell lines upon inhibition of the proteasome (MG132 treated samples) demonstrating that Hit (#<NUM>) and Hit (#<NUM>) lead to selective GFP degradation via functional recruitment of E3 ligases and the Ubiquitin Proteasome System, meaning Hit (#<NUM>) and Hit (#<NUM>) are E3-binding peptides (degrons).

High confidence hits from a screen (candidate peptides that led to selective degradation of the test protein under conditions enabling ubiquitination of proteins by an E3, i.e. degron peptides) were clustered by amino acid sequence similarity using Clustal Omega (https://www. uk/Tools/msa/clustalo/). The graph (<FIG>) shows clusters of identical amino acid sequences being enriched in the hit pool, indicative of families of common motifs among the hits able to facilitate functional recruitment of E3 ligases to a substrate. These common motifs may correlate with particular interacting E3s. Further, the common motifs can be enriched and combined in further rounds of candidate peptide generation and screening in order to increase the efficiency and/or specificity of degradation via E3s.

Host cells (HEK293T) were infected with a lentivirus mixture comprising the full DegroPEx library of 46AA peptides fused to GFP or an "empty" reporter-only construct. Cells were then treated with either vehicle (DMSO), cycloheximide (CHX), MG132, or both MG132 and CHX. After four hours, the cells were analysed by flow cytometry to evaluate the fluorescence rate of each treatment group.

Vehicle-treated reporter-only cells were found to be highly fluorescent (Figure 11A-C, dark trace), whilst library-infected vehicle-treated cell equivalents showed a distinct low-fluorescence population (Figure 11D-F, dark trace).

Treatment of the library-infected group with the proteasome inhibitor MG132 reverted it to a more fluorescent signature (Figure 11E, light trace), whilst treatment with translation blocker cycloheximide (CHX) caused a decrease in the overall fluorescence signature (Figure 11A, 11D, light trace), shifting the hit population to a very low fluorescence measure (Fig. 11D, light trace). Treatment of the library-infected group with both MG132 and CHX (Fig. 11F) resulted in a trace similar to that for MG132 treatment alone.

These results indicate that the cause of fluorescence decrease in the peptide hits is not a block in translation, since CHX treatment alone results in further diminishment of fluorescence. This demonstrates that the low reporter signal in hits is caused by degradation of the protein via the UPSsystem.

Nominated hit peptide sequences from both an 11AA and 46AA screen generated according to Example <NUM> were resynthesized and recloned as individual homogeneous plasmids and used to generate new reporter-peptide hybrids in individual lentivirus reagents. These were used to infect new populations of cells which, following selection, were evaluated for the fluorescence signature as described above. This was reported as the modal fluorescence intensity and normalized to the reporter-only construct (<FIG>, 'GFP_CTRL', intensity of <NUM>)). Individual hits were successfully validated from both primary screens where they gave a normalized modal intensity of less than <NUM>. For both screens, A von Hippel-Lindau (VHL) ligand fused to the reporter was used as a positive control, in each case giving a normalized modal intensity of less than <NUM> as expected (<FIG>).

Performing one-by-one validation of hits in this way allowed refined quantitative ranking of validated hits, as the normalized modal intensity of each could be compared.

Following further treatment with proteasome inhibitor MG132 (data not shown), all hits showed a reversal of the effect on fluorescence, indicating that the reduction in fluorescence was due to degradation of the reporter protein via the ubiquitin proteasome system recruited by the hit peptide.

Five hit peptide sequences identified from screens as described above were each fused to the Tau protein isoform Tau441 sequence (instead of the GFP reporter sequence), and were then introduced into cells by transient transfection. Tau441 is a disease-associated long protein isoform, which in neurons of affected patients can aggregate and cause or potentiate neurodegenerative illnesses. It is approximately twice the length of GFP, with very different physicochemical properties, including but not limited to the absence of fluorescence and propensity to aggregate as described above. Following incubation for <NUM> hours, total protein was extracted from the cells and separated by gel electrophoresis. Degradation of Tau was analysed by performing western blotting (<FIG>) using an anti-Tau antibody (ab32057; AbCam), compared with the expression level of the housekeeping gene GAPDH (<NUM>; Cell Signalling Technologies). Tau441 alone (without a fused peptide) was used as a degradation-negative control ('CTRL' on <FIG>), and Tau441 fused to a VHL ligand was used as a degradation-positive control ('CTRL-VHL' on <FIG>).

Total Tau protein was significantly diminished compared to GAPDH when Tau was fused to one of three screen hit peptides (A2, E4. This demonstrates that these three peptides can direct the proteasomal degradation of the diverse proteins GFP and Tau.

When bound to Tau the other two GFP-screen derived peptides tested (E10, I7) showed similar levels of Tau protein to that of GAPDH, indicating that those peptides induced reduced degradation of Tau in comparison with peptides A2, E4 and H8. That was also the case forTau bound to a VHL ligand (`CTRL-VHL'). This indicates that some of the novel peptide sequences, and VHL, elicit a substrate-specific response.

Selected hit peptides A2 and E4 were individually recloned as GFP fusions and used to generate homogenous cell populations. The cell populations, plus cell populations expressing either untagged GFP reporter or GFP fused to a VHL ligand, were incubated with and without the presence of the proteasomal inhibitor MG132. Following a <NUM>-hour incubation , whole cell lysates were prepared. Immunoprecipitations (IP) were conducted using an anti-GFP antibody (<NUM>; Cell Signalling Technologies) to purify the reporter protein and reporter-peptide fusion proteins from the whole-cell lysates. Western blotting (WB) was then used to probe for ubiquitin bound to the reporter or to the peptide-reporter fusion proteins, using either an anti-ubiquitin antibody (<NUM>; clone P4D1; Cell Signalling Technologies), or an anti-K48 linkage-specific ubiquitin antibody (<NUM>; clone D9D5; Cell Signalling Technologies), or to probe for GFP using an anti-GFP antibody (<NUM>; Cell Signalling Technologies).

See <FIG> for results. As expected, reporter expressed without a peptide tag showed no ubiquitination and the greatest levels of GFP in the Western blot. The presence of MG132 during cell incubation enhanced the ubiquitination levels observed with the hit peptide-GFP fusion proteins, using either the anti-ubiquitin or anti-K48 linkage specific antibody to probe the Western blots. The hit peptide-GFP fusions showed K48 (degron-specific) ubiquitination, to varying extents.

Claim 1:
A method for determining if a peptide binds or is capable of binding to a ubiquitin protein ligase (E3) and thereby leads to degradation of a test protein, wherein the peptide is between about <NUM> and <NUM> amino acids in length, the method comprising:
- providing in a eukaryotic cell a candidate peptide functionally linked to a test protein, under conditions enabling ubiquitination of proteins by an E3; and
- detecting the amount of test protein present in the cell;
whereby, a reduced amount of the test protein determines the candidate peptide as a peptide that binds or is capable of binding to an E3 (an E3-binding peptide);
wherein the candidate peptide is functionally linked to the test protein and the candidate peptide is comprised in at least one hybrid polypeptide as a domain; wherein the hybrid polypeptide comprises a domain being a test protein or a test protein binding (poly)peptide; and a domain being a peptide linker;
and wherein the amino acid sequence of the test protein is genetically engineered to comprise a plurality of lysine residues, wherein the test protein is genetically engineered to comprise an N-terminal lysine-rich region derived from human nitric oxide synthase (NOS1); and
wherein the candidate peptide functionally linked to the test protein is provided as a member of a library of candidate peptides, suitably each functionally linked to the test protein; and
wherein the candidate peptides comprised in the library have a length of between about <NUM> and <NUM> amino acids, and have an amino acid sequence being a region of a sequence selected from the amino acid sequence of a naturally occurring protein of one or more organisms; wherein the library comprises a plurality of at least <NUM>,<NUM> different such peptides, and wherein the amino acid sequence of each of at least <NUM> of such peptides is a sequence region of the amino acid sequence of a different protein of a plurality of different such naturally occurring proteins.