Fusion proteins containing E2 ubiquitin or ubiquitin-like conjugation domains and targeting the domains for specific protein degradation

Abstract

The present disclosure provides molecules comprising a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a human E2 ubiquitin or ubiquitin-like domain, and a targeting domain capable of targeting the regulatory domain to a substrate. Also provided are polynucleotides encoding such molecules, methods for identifying and producing such molecules, and related pharmaceutical compositions and kits suitable for use, particularly in treating or preventing diseases and / or conditions in a subject that are mediated by dysregulated substrates.

Description

[Background technology]

[0001] Ubiquitination is characterized by the rapid and reversible post-translational covalent attachment of ubiquitin to proteins. This mechanism plays an important role in targeting proteins for degradation and regulating their subcellular localization, intracellular signaling, and interactions with other proteins (Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3).

[0002] Ubiquitin (Ub) is a small (76 amino acids; 8.6 kDa) regulatory protein. The addition of ubiquitin to a protein is called ubiquitination. Ubiquitination involves activation of the ubiquitin C-terminus. For this to occur, an E1 Ub-activating enzyme forms a thioester bond with Ub in an adenosine triphosphate-dependent reaction. This is followed by conjugation to an E2 ubiquitin-conjugating enzyme (and potentially a HECT-type E3 ubiquitin ligase as an E3-Ub intermediate) and ligation to the substrate protein. Ubiquitin can be attached to target substrates through (i) lysine residues via an isopeptide bond, (ii) cysteine ​​residues via a thioester bond, (iii) serine and threonine residues via an ester bond, or (iv) the amino group at the N-terminus of the protein via a peptide bond.

[0003] Either a single ubiquitin protein (monoubiquitination) or ubiquitin chains (polyubiquitination) can be attached to a substrate. In polyubiquitin chains, a second ubiquitin molecule is attached to one of the seven lysine residues (e.g., K48 or K63) or the N-terminal methionine of the previous ubiquitin molecule. Attachment of ubiquitin to proteins allows for protein regulation by marking them for degradation by the proteasome, changing their cellular location, affecting their activity, and promoting or preventing protein interactions.

[0004] Several proteins similar to ubiquitin, called ubiquitin-like proteins (Ubls), can be used in a similar mechanism. The human genome encodes at least eight families of ubiquitin-like proteins, none of which include ubiquitin itself, which are considered type I Ubls. These are small ubiquitin-like modifier (SUMO), developmentally downregulated neural precursor 8 (NEDD8), autophagy-related protein 8 (ATG8), autophagy-related protein 12 (ATG12), ubiquitin-related modifier 1 (URM1), ubiquitin-folding modifier 1 (UFM1), ubiquitin-like protein FAT10, and interferon-stimulated gene 15 (ISG15). Type I Ubls are capable of covalent conjugation. Covalent conjugation occurs via one or two glycine residues at the C-terminus. Humans also encode a type 2 Ubl, Fau ubiquitin-like protein (FUBI), which is incapable of covalent conjugation. Although proteins tagged with SUMO or NEDD8 are not recognized for degradation, they play roles in gene transcription activation, protein localization, and stabilization. Each target function combination has a unique combination of E1, E2, and E3 enzymes.

[0005] E2 enzymes act to transfer ubiquitin to target substrates, and all share a core catalytic domain of approximately 150 amino acids called the ubiquitin core catalytic domain (UBC domain). Generally, the domain typically adopts an α / β fold, with four α-helices and a four-stranded β-sheet (Non-Patent Document 4). A critical loop region forms part of the E3 binding site and the E2 active site. This surface is involved in binding to both the RING-E3 domain and the HECT-E3 domain and overlaps with the region recognized by E1 enzymes. The UBC domain is approximately 14-16 kDa and is approximately 35% conserved among different family members (Non-Patent Document 5). E2s have a consensus fold adapted to specific systems. While most E2s contain only a single structural UBC domain, many possess short N- and / or C-terminal extensions that may confer important E2-specific functionality, such as recognition of bound ubiquitin to chain-assembling E2s. Some E2s, including Ube2R1 and Ube2G2, have functionally important insertions, and some E2s have additional structural domains linked to the UBC domain (e.g., Ube2K) or are part of large multidomain proteins (Ube2O or BIRC6).

[0006] E2 enzymes are primarily involved in two types of reactions for the transfer of ubiquitin from the E2-Ub conjugate to the substrate: (1) transthiolation (transfer of thioester to thiol group, e.g., transfer of ubiquitin to the active site cysteine ​​residue of HECT-type E3 ligases) and (2) aminolysis (transfer of thioester to amino group), although others have also been reported (Non-Patent Document 4).

[0007] All E2s interact with E1 enzymes and one or more E3s. Furthermore, E2s can directly engage target proteins (e.g., so-called E3 / E2 hybrids, such as BIRC6 and UBE2O, are E3-independent because they can interact with and ubiquitinate their substrates without assistance from an E3), and thus may play a role in determining where and how targets are modified by ubiquitin. To date, there are no examples of E3s altering the chemical reactivity profile of E2s, so the intrinsic reactivity of a given E2 likely predicts the nature of its products.

[0008] Based on their mechanistic strategies, E3s have been classified into three families: RING, HECT, and RING-between-RINGS (RBR). RING / U-box E3 ubiquitin ligases can bind both substrates and E2-Ub conjugates. HECT / RBR domain E3 ligases must also be able to form an intermediate thioester with ubiquitin (E3-Ub). Some E2s can function with multiple types of E3s.

[0009] HECT domain-containing E3 ubiquitin ligases form an intermediate thioester bearing Ub at their active site cysteine ​​(E3~Ub) before transferring Ub to the substrate, whereas most RING finger domain-containing E3 enzymes act as a scaffold to simultaneously bind the E2 enzyme and the substrate.

[0010] RING E3s (most E3s) are not directly involved in the chemical transfer of Ub to the substrate. They bind to the substrate and the E2-Ub conjugate, facilitating the direct transfer of Ub from the E2 active site to the substrate. RING E3s function as protein cofactors for the E2-Ub conjugate. Although the RING E3 / E2-Ub complex is dynamic, interaction with a RING E3 increases the intrinsic reactivity of many (but not all) E2-Ub conjugates toward aminolysis. For example, the Ube2D family of E2s reacts slowly with lysines in the absence of an E3 but rapidly in the presence of a RING domain.

[0011] E2~Ub generally adopts a "closed state" upon RING E3 binding. A conserved RING (allosteric linking pin) residue, usually arginine, lysine, or asparagine, donates a hydrogen bond to the E2 backbone carbonyl in loop 7 and one or more backbone groups in the Ub tail. This E2~Ub closed state is considered the active state for aminolysis. The transthiolation reaction can readily occur in the absence of an E3. Therefore, it is hypothesized that an E3 proceeding on the E3~Ub conjugated intermediate (e.g., HECTE3s) is not required to promote the E2~Ub closed state.

[0012] It will be appreciated that the ability to regulate specific targets through their modification by ubiquitin or ubiquitin-like proteins has potential utility in studying protein function and in combating disease.

[0013] PROteolysis-TArgeting Chimeras (PROTACs) are engineered chemical entities that utilize the ubiquitin-proteasome pathway to enable the temporally controlled removal of proteins in a post-translational manner through simultaneous binding of target proteins to E3 ligases. PROTAC molecules contact target proteins with E3 ubiquitin ligases, promoting the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes, resulting in the ubiquitination and proteasomal degradation of the target protein. PROTACs have great potential for targeting previously "undruggable" proteins for use in drug discovery and the development of new therapeutics (Non-Patent Document 6). The first generation of PROTACs was a peptide-based PROTAC containing a phosphopeptide that binds to the E3 ligase β-TRCP and the small molecule ovalicin, which targets MetAP-2 (Non-Patent Document 6). Since then, small molecule PROTACs, MDM2-based PROTACs, IAP-based PROTACs, CRBN-based PROTACs, and VHL-based PROTACs have been developed. For example, over 30 small molecule PROTACs have been reported that target the androgen receptor (Non-Patent Document 7), cyclin-dependent kinase 9 (Non-Patent Document 8 and Non-Patent Document 9), and c-Met, and degradation of targeted proteins offers several advantages over inhibition in terms of potency, selectivity, and drug resistance (Non-Patent Document 10).

[0014] A series of biological PROTACs have also been developed. For example, Non-Patent Document 11 describes so-called ubiquibodies, engineered protein chimeras that combine the activity of E3 ubiquitin ligases with designer binding proteins such as single-chain Fv intrabodies or fibronectin type III domain (FN3) monobodies. Non-Patent Document 12 developed a recombinant chimeric protein that specifically induces the degradation of mutant KRAS and potently inhibits pancreatic tumor growth. The chimeric protein contains the Ras-binding domain (RBD) of Raf1 and an E3 adaptor protein. Non-Patent Document 13 describes an affinity-directed protein missile (AdPROM system) that carries the von Hippel-Lindau (VHL) protein, a substrate receptor for the Cullin2 (CUL2) E3 ligase complex, tethered to a polypeptide binder that selectively binds and recruits endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. Another biologically based degradation system is the so-called Trim-Away technology developed by

[14] , which involves TRIM21, an E3 ubiquitin ligase that binds with high affinity to the Fc domain of antibodies.

[0015] However, before PROTAC technology can be matured for clinical use, various problems remain, such as off-target effects, in vivo metabolic stability, cell permeability, and high molecular weight. The synthesis and optimization of such bifunctional molecules are also difficult, which are significant obstacles in research and manufacturing. Therefore, there is still a need for more such bifunctional molecules. [Prior art documents] [Non-patent literature]

[0016] [Non-Patent Document 1] Glickman and Ciechanover, Physiol Rev 2002,82(2):373-428 [Non-patent document 2] Mukhopadhyay and Riezman,Science 2007,315(5809):201-5 [Non-licensed document 3] Schnell and Hicke,J Biol Chem 2003,278(38):35857-60

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Non-licensed Document 7

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Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0017] Despite all known biological PROTACs requiring E3 ubiquitin ligases, the inventors have surprisingly and unexpectedly identified a novel class of molecules containing E2 enzymes that can provide targeted degradation or regulation of proteins via the ubiquitin or ubiquitin-like protein pathway. Such molecules are simpler to produce, smaller, easier to deliver, less dependent on endogenous proteins, easier to regulate, and can reach a broader set of targets.

[0018] Provided herein are molecules comprising (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a human E2 enzyme or a functional portion thereof, and (b) a targeting domain capable of targeting the regulatory domain to a substrate. In certain embodiments, the molecule does not comprise an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof. In some embodiments, the molecule is a fusion polypeptide. In some embodiments, the regulatory domain is N-terminal to the targeting domain. In other embodiments, the regulatory domain is C-terminal to the targeting domain.

[0019] Further provided herein is a compound comprising the aforementioned molecule and a targeting moiety capable of targeting the molecule to a cell. Further provided herein is the use of a compound comprising (i) the aforementioned molecule and (ii) a targeting moiety capable of targeting the molecule to a cell in the manufacture of a medicament for delivering a molecule in an individual.

[0020] The present disclosure further provides polynucleotides encoding the aforementioned molecules or compounds. Further provided herein are vectors comprising the aforementioned polynucleotides, such as adeno-associated viral (AAV) vectors or lentiviral vectors. Further provided herein are host cells comprising the polynucleotides or vectors. Further provided herein are compositions comprising the aforementioned molecules or compounds and an additional therapeutic agent.

[0021] Further provided herein is a pharmaceutical composition comprising the aforementioned molecule, the aforementioned compound, the aforementioned polynucleotide, the aforementioned vector, the aforementioned host cell, or the aforementioned composition, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0022] Another aspect of the present disclosure provides a method of delivering the aforementioned molecule to a cell of an individual, the method comprising administering to the individual (i) the molecule and (ii) a compound comprising a targeting moiety capable of targeting the molecule to the cell, or administering to the individual the aforementioned polynucleotide or vector, wherein the polynucleotide or vector encodes the molecule in the cell.

[0023] Another aspect of the present disclosure provides a kit-of-parts comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; and (b) a targeting domain capable of targeting the regulatory domain to a substrate; optionally, the kit does not comprise an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof.

[0024] Further provided herein is a kit-of-parts comprising (a) the aforementioned molecule and (b) a targeting moiety capable of targeting a cell containing the substrate to be modulated, optionally wherein the targeting moiety is a binding partner such as an antibody. Further provided herein is a method of preventing or treating a disease or condition in a subject mediated by abnormal levels of a substrate or a form thereof, the method comprising administering to the subject the above-described molecule, compound, polynucleotide, vector, host cell, pharmaceutical composition, or composition. Further provided herein is the above-described molecule, compound, polynucleotide, vector, host cell, pharmaceutical composition, or composition for use in preventing or treating a disease or condition mediated by abnormal levels of a substrate or a form thereof in a subject. In some embodiments, the disease or condition is cancer, diabetes, an autoimmune disease, Alzheimer's disease, Parkinson's disease, pain, a viral disease, a bacterial disease, a prion disease, a fungal disease, a parasitic disease, arthritis, an immune deficiency, or an inflammatory disease.

[0025] Further provided herein are methods of modulating a substrate, comprising contacting the substrate with a molecule under conditions effective for the molecule to modulate the substrate. In some embodiments, modulating comprises degrading the substrate, or preventing the substrate from being degraded, or altering the intracellular location of the substrate, or modulating (e.g., increasing or decreasing) one or more activities of the substrate, or modulating the degree of post-translational modification of the substrate.

[0026] Further provided herein is a method for identifying a substrate as a potential drug target, the method comprising: (a) providing a cell, tissue, or organ comprising the substrate; (b) contacting the cell, tissue, or organ with the molecule, compound, polynucleotide, or vector; and (c) assessing the effect of the molecule, compound, polynucleotide, or vector on one or more properties of the cell, tissue, or organ, wherein identification of an effect that correlates with a particular disease state indicates that the substrate is a potential drug target for the particular disease.

[0027] Further provided herein is a method of assessing the function of a substrate, the method comprising: (a) providing a cell, tissue, or organ comprising the substrate; (b) contacting the cell, tissue, or organ with the molecule, compound, polynucleotide, or vector; and (c) assessing the effect of the molecule, compound, polynucleotide, or vector on one or more properties of the cell, tissue, or organ.

[0028] Further provided herein are methods for identifying a test agent that may be useful in preventing or treating a disease or condition mediated by abnormal levels of a substrate or a form thereof, the method comprising: providing the substrate; and providing a test agent comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence having at least 80% sequence identity to a human E2 ubiquitin or ubiquitin-like domain; and (b) a targeting domain capable of targeting the regulatory domain to the substrate, wherein optionally the test agent does not comprise an E3 ubiquitin or ubiquitin-like ligase or portion thereof; contacting the substrate and the test agent under conditions effective for the test agent to facilitate modulation of the substrate; and determining whether the test agent modulates the substrate. In some embodiments, the method further comprises testing the test agent in an assay for the disease or condition. [Brief explanation of the drawings]

[0029] [Figure 1]Figure 1A shows a Western blot of MDA-MB-231 cell lysates after lentiviral transduction of the encoded control and fusion polypeptide constructs. SHP2 and loading control alpha-tubulin are shown. Two replicate samples of the E2 fusion polypeptide UBE2D1_aCS3, with reduced SHP2 protein levels, are shown. Black boxes identify lysates from cells transduced with the E3 ligase fusion polypeptide, and gray boxes identify the E2 fusion polypeptide construct. Figure 1B shows a graph depicting densitometry of the Western blot signal. Band densities for SHP2 protein levels were normalized to the alpha-tubulin loading control band densities and then expressed as a percentage of control MDA-MB-231 cell SHP2 levels. Data represent multiple replicates in multiple cell lines. [Figure 2] Degradation of SHP2 protein in MDA-MB-231 cells comparing the orientation and linker length of E3 ligase and E2 biological fusion polypeptides. Figure 2A shows a Western blot of MDA-MB-231 cell lysates after lentiviral transduction of the encoded control and fusion polypeptide constructs. SHP2 protein and GAPDH loading controls are shown. Black boxes identify lysates from cells transduced with E3 ligase fusion polypeptides, and gray boxes identify E2 fusion polypeptide constructs. Figure 2B shows a graph depicting densitometry of the Western blot signals. Band densities for SHP2 protein levels were normalized to GAPDH loading control band densities and then expressed as a percentage of control MDA-MB-231 cell SHP2 levels. The UBE2D1 regulatory domain construct uses the shorter name E2D1. Within the sample names, "short" and "long" refer to different linker lengths of 9 and 19 amino acids, respectively. [Figure 3]Figure 3A shows degradation of SHP2 protein in U2OS cells, comparing the orientation and linker length of E3 ligase and E2 biological fusion polypeptides. Figure 3A shows Western blots of U2OS cell lysates after lentiviral transduction of the encoded control and fusion polypeptide constructs. SHP2 protein and GAPDH loading controls are shown. Black boxes identify lysates from cells transduced with E3 ligase fusion polypeptides, and gray boxes identify E2 fusion polypeptide constructs. Figure 3B shows densitometry of the Western blot signals. Band densities of SHP2 protein levels were normalized to GAPDH loading control band densities and then expressed as a percentage of control U2OS cell SHP2 levels. The UBE2D1 regulatory domain construct uses the shorter name E2D1. Within the sample names, "short" and "long" refer to different linker lengths of 9 and 19 amino acids, respectively. Figure 3C is a graph comparing different linker lengths and the efficiency of SHP2 degradation using the E2D1_Linker_aCS3 (UBE2D1_Linker_aCS3) construct based on the fluorescent signal after imaging with Cytation5 (Biotek®). Fluorescence intensities of SHP2 and HA-tagged protein levels were probed using antibodies specific for these epitopes, and SHP2 levels were normalized to a range of 0-100% based on the SHP2 levels found in untreated cells within each experiment. Data correspond to n = 3 or more biological replicates. The number of residues in the linker length refers to the number of amino acids in the linker sequence. [Figure 4]Figure 4A shows degradation of SHP2 protein in MDA-MB-231 cells, comparing high- and low-affinity variants of SHP2-binding monobodies as binding domains. The standard aCS3 monobody, which has high affinity for SHP2 (SHP2 C-SH2 domain Kd = 4-9.1 nM), was compared with the V33R aCS3 mutant, which has lower affinity (SHP2 C-SH2 domain Kd = 1.2 μM). (Sha et al., Proc. Natl. Acad. Sci. US Patent Application Publication No. 2013110(37):14924-9 and supplementary information). Note: The monobody aCS3 is also referred to as CS3 herein. Figure 4B shows Western blots of MDA-MB-231 cell lysates after lentiviral transduction of encoded control and fusion polypeptide constructs containing the aCS3 and aCS3 V33R mutant binding domains. SHP2 protein and GAPDH loading controls are shown. Black boxes identify lysates from cells transduced with the E3 ligase fusion polypeptide, and gray boxes identify the E2 fusion polypeptide construct. Two replicate samples of E2D1_long_aCS3 are present on the Western blot. Figure 4B is a graph showing densitometry of the Western blot signal. Band densities of SHP2 protein levels were normalized to GAPDH loading control band densities and then expressed as a percentage of control MDA-MB-231 cell SHP2 levels. The UBE2D1 regulatory domain construct uses the shorter name E2D1. In the sample names, "long" refers to the 19-amino acid linker between the regulatory and binding domains. [Figure 5]Figure 5A shows degradation of SHP2 protein in U2OS cells comparing high- and low-affinity variants of SHP2-binding monobodies as binding domains. The standard aCS3 monobody, which has high affinity for SHP2 (SHP2 C-SH2 domain Kd = 4-9.1 nM), was compared with the V33R aCS3 mutant, which has lower affinity (SHP2 C-SH2 domain Kd = 1.2 μM). (Sha et al., Proc. Natl. Acad. Sci. US Patent Application Publication No. 2013110(37):14924-9 and supplementary information). Note: The monobody aCS3 is also referred to as CS3 herein. Figure 5B shows Western blots of U2OS cell lysates after lentiviral transduction of encoded control and fusion polypeptide constructs containing the aCS3 and aCS3 V33R mutant binding domains. SHP2 protein and GAPDH loading controls are shown. Black boxes identify lysates from cells transduced with E3 ligase fusion polypeptides, and gray boxes identify E2 fusion polypeptide constructs. Figure 5B is a graph showing densitometry of Western blot signals. Band densities of SHP2 protein levels were normalized to GAPDH loading control band densities and then expressed as a percentage of control U2OS cell SHP2 levels. The UBE2D1 regulatory domain construct uses the shorter name E2D1. In the sample names, "long" refers to the 19-amino acid linker between the regulatory and binding domains. [Figure 6]Figure 6 shows a comparison of KRas degradation using K19 DARPin_E2 and K19 DARPin_E3 fusion polypeptides. DARPin K19 binds both GDP- and GTP-bound KRAS (Bery et al., Nat Commun 2019 10(1):2607), while E3_5 is a negative control (non-binding) DARPin. DARPin fusion polypeptide constructs were tested in both MDA-MB-231 and Ad293 cell lines. Figure 6A shows Western blots of MDA-MB-231 and Ad293 cell lysates after lentiviral transduction of encoded control and fusion polypeptide constructs. KRas protein and α-tubulin loading control protein are shown. Black boxes identify lysates from cells transduced with E3 ligase fusion polypeptides, and gray boxes identify E2 fusion polypeptide constructs. Figure 6B shows a graph showing densitometry of the Western blot signals. The band density of KRAS protein levels was normalized to the α-tubulin loading control band density and then expressed as a percentage of control MDA-MB-231 or Ad293 cell KRAS levels, respectively. [Figure 7]Schematic diagram of E3 and E2 fusion polypeptides. Figure 7A shows an example of an E3 biological fusion polypeptide containing the E3 ligase VHL fused to a binding domain via a linker, as previously described by Fulcher et al. (Fulcher et al., 2017, Open Biol 7:170066). The binding domain (e.g., a monobody, nanobody, or antibody mimetic) can recruit a target protein within a cell (either an endogenous protein or a non-endogenously or ectopically expressed protein, such as a viral protein) to the EloB / C / CUL2 / RBX1 E3 ligase machinery. This complex then binds to an E2 conjugating enzyme, which enables the transfer of ubiquitin to the target protein. The addition of multiple ubiquitin molecules to form a chain, known as polyubiquitination, tags the target protein for degradation by the proteasome. Alternative E3 ligase fusion polypeptides may require the involvement of different proteins to enable ubiquitination of the target protein. Figure 7B is a diagram of an example of an E2 biological fusion polypeptide containing an E2 ubiquitin conjugation domain fused directly to a conjugation domain via a linker. The conjugation domain (e.g., a monobody, nanobody, or antibody mimetic) can bind to a target protein (an endogenous protein or a non-endogenously or ectopically expressed protein, such as a viral protein) within a cell, allowing the E2 ubiquitin conjugation domain to transfer ubiquitin to the target protein. Polyubiquitination of the target protein leads to degradation of the target protein by the proteasome. In these examples, if the E2 ubiquitin conjugation domain were replaced with a ubiquitin-like conjugation domain, a ubiquitin-like molecule would be transferred to the target protein (e.g., SUMO, NEDD8, RUB1, ATG8, ATG12, ISG15, FAU, or URM1) rather than ubiquitin being transferred to the target protein. [Figure 8]Figure 8A shows an investigation of the effect of a panel of E2 ubiquitin-conjugating enzymes and E2 ubiquitin-like conjugating enzyme core domains fused to the aCS3 conjugating domain on SHP2 protein expression in MDA-MB-231 cells. Twenty-six different E2 ubiquitin-conjugating enzymes and E2 ubiquitin-like conjugating enzyme core domains were encoded as fusion proteins on a lentiviral plasmid in the format HA tag_E2_Linker_aCS3. Lentiviral particles were then generated and used to transduce MDA-MB-231 cells. Figure 8B shows a Western blot of MDA-MB-231 cell lysates after lentiviral transduction of the encoded control and E2 fusion constructs. The Western blot was probed with antibodies against SHP2, the HA tag (indicating the expression levels of the fusion proteins), and alpha-tubulin (as a loading control). Protein lysates from MDA-MB-231 cells transduced with lentiviral particles encoding the UBE2D1_aCS3 fusion protein were run on separate gels for comparison purposes. Figure 8B is a graph showing the amount of SHP2 protein observed with each E2 core domain fusion protein compared to the SHP2 degradation observed with the UBE2D1_aCS3 fusion polypeptide. Values ​​were calculated using densitometry of the Western blot signal. Band densities for SHP2 protein levels were normalized to the band densities of the α-tubulin loading control and then expressed as a percentage of the SHP2 levels observed for cells transduced with lentiviral particles encoding the UBE2D1_aCS3 fusion polypeptide. Core domains of interest were those that could reduce SHP2 protein levels to a similar or greater extent than UBE2D1_aCS3. [Figure 9]Figure 9A shows an investigation of the effect of a panel of E2 ubiquitin-conjugating enzymes and E2 ubiquitin-like conjugating enzyme core domains fused to the aCS3 conjugating domain on SHP2 protein expression in U2OS cells. Twenty-six different E2 ubiquitin-conjugating enzymes and E2 ubiquitin-like conjugating enzyme core domains were encoded as fusion proteins on a lentiviral plasmid in the format HA-tag_E2_Linker_aCS3. Lentiviral particles were then generated and used to transduce U2OS cells. Figure 9B shows a Western blot of U2OS cell lysates after lentiviral transduction of the encoded control and E2 fusion constructs. The Western blot was probed with antibodies against SHP2, the HA tag (indicating the expression level of the fusion protein), and alpha-tubulin (as a loading control). Protein lysates from U2OS cells transduced with lentiviral particles encoding the UBE2D1_aCS3 fusion protein were run on separate gels for comparison purposes. Figure 9B is a graph showing the amount of SHP2 protein observed with each E2 core domain fusion protein compared to the SHP2 degradation observed with the UBE2D1_aCS3 fusion polypeptide. Values ​​were calculated using densitometry of the Western blot signals. Band densities of SHP2 protein levels were normalized to the band densities of α-tubulin loading controls and then expressed as a percentage of the SHP2 levels observed for cells transduced with lentiviral particles encoding the UBE2D1_aCS3 fusion polypeptide. Core domains of interest were those that were able to reduce SHP2 protein levels to a similar or greater extent than UBE2D1_aCS3. [Figure 10]This figure shows the effect of mutations of lysine residues within the aCS3 binding domain on SHP2 degradation and the expression level of the fusion polypeptide. The aCS3 monobody contains three lysine residues (K7, K55, and K64) and is prone to (self-)ubiquitination and degradation when expressed as a fusion protein with an E2 ubiquitin-conjugating enzyme. The three aCS3 lysine residues were mutated individually and in combination and then expressed in cells as the binding domain of a UBE2D1 fusion polypeptide in the HA tag_E2D1_Linker_aCS3 format. In-house structural modeling indicated which amino acid residue changes should maintain monobody stability. Lysine residue K7 was mutated to glutamine (K7Q). Lysine residue K55 was mutated to tyrosine (K55Y), and lysine residue K64 was mutated to histidine (K64H). The effects on SHP2 degradation and fusion polypeptide expression in cells expressing fusion polypeptides containing these aCS3 variants were measured by Western blot probing for SHP2 protein and HA tag expression levels, respectively. Figure 10A shows Western blots of U2OS cell lysates after lentiviral transduction of the encoded control and lysine-mutated aCS3 variant fusion polypeptide constructs. The Western blots were probed with antibodies against SHP2, the HA tag (indicating the expression levels of the fusion protein), and alpha-tubulin (as a loading control). Figure 10B shows a graph depicting densitometry of the Western blot signals. The band density of the SHP2 protein levels was normalized to the alpha-tubulin loading control band density and then expressed as a percentage of the control U2OS cell SHP2 levels. Figure 10C shows a graph depicting densitometry of the Western blot signals. The band density of the HA-tagged fusion polypeptide levels was normalized to the band density of the α-tubulin loading control and then expressed as a percentage of the HA-tagged UBE2D1_aCS3(WT) levels. Figure 10 continues. Mutating the catalytic site of the UBE2D1 or UBE2B regulatory domain of the fusion polypeptide or reducing the binding domain affinity for the target protein reduces target protein degradation.Figure 10D shows that U20S cells were transfected with mRNA encoding variants of the SHP2-targeting fusion polypeptide (using the aCS3-binding domain with all lysines removed, i.e., K7Q, K55Y, and K64H), and target SHP2 protein levels were determined by Western blotting after 24 hours of incubation. U20S cells were transfected with mRNA encoding EGFP (non-degraded mRNA) as a control. The variants included (i) mutating the catalytic cysteine ​​residues in regulatory domains, e.g., UBE2D1 (C85A) and UBE2B (C88A), (ii) reducing the binding domain affinity for SHP2 with the V33R mutation in aCS3, and (iii) mutating a UBE2D1 residue involved in interaction with the E3 ligase (i.e., F62A) to determine the effect on activity. Figure 10E shows quantification of SHP2 expression levels from densitometric measurements of Western blot band intensities relative to loading control levels and normalization to SHP2 levels in U20S cells transfected with EGFP for UBE2D1 fusion polyproteins, and Figure 10F shows quantification of SHP2 expression levels from densitometric measurements of Western blot band intensities relative to loading control levels and normalization to SHP2 levels in U20S cells transfected with EGFP for UBE2D1 fusion polyproteins, and for UBE2B fusion polyproteins. [Figure 11]Figure 11A shows the degradation of human antigen receptor (HuR) in MDA-MB-231 cells using UBE2D1 fusions containing HuR-targeting VHH nanobody (HuR8 and HuR17) binding domains. HuR is primarily a nuclear protein. A control UBE2D1 fusion protein with a Cas9 VHH nanobody binding domain is included. Cas9 is a bacterial protein and therefore not endogenously expressed in mammalian cells. Therefore, Cas9 VHH nanobodies should not selectively bind to any proteins in mammalian cells. The effect on HuR protein levels was investigated for both orientations of the fusion construct. Figure 11B shows a Western blot of MDA-MB-231 cell lysates after lentiviral transduction of the encoded control (UBE2D1_Cas9VHH) and HuR-binding variant fusion polypeptide constructs, UBE2D1_HuR17 and UBE2D1_HuR8. Western blots were probed with antibodies against HuR and alpha-tubulin (as loading controls). Figure 11B is a graph showing densitometry of the Western blot signals. Band densities of HuR protein levels were normalized to the alpha-tubulin loading control band densities and then expressed as a percentage of the HuR levels observed for cells expressing UBE2D1_Cas9VHH. Figure 11C is a Western blot of MDA-MB-231 cell lysates after lentiviral transduction of the encoded control (Cas9VHH_UBE2D1) and HuR-binding variant fusion polypeptide constructs, HuR17_UBE2D1 and HuR8_UBE2D1. Western blots were probed with antibodies against HuR and alpha-tubulin (as loading controls). Figure 11D is a graph showing densitometry of the Western blot signals. The band density of HuR protein levels was normalized to the α-tubulin loading control band density and then expressed as a percentage of the HuR levels observed for cells expressing Cas9VHH_UBE2D1. [Figure 12]Figure 12A shows the investigation of human antigen receptor (HuR) degradation in U20S cells using UBE2D1 fusions containing HuR-targeting VHH nanobody (HuR8 and HuR17) binding domains. HuR is primarily a nuclear protein. A control UBE2D1 fusion protein with a Cas9 VHH nanobody binding domain was included. Cas9 is a bacterial protein and is therefore not endogenously expressed in mammalian cells. Therefore, Cas9 VHH nanobodies should not selectively bind to any proteins in mammalian cells. The effect on HuR protein levels was investigated for both fusion construct orientations. Figure 12B shows a Western blot of U20S cell lysates after lentiviral transduction of the encoded control (UBE2D1_Cas9VHH) and HuR-binding variant fusion polypeptide constructs, UBE2D1_HuR17 and UBE2D1_HuR8. The Western blot was probed with antibodies against HuR and alpha-tubulin (as loading controls). Figure 12B is a graph showing densitometry of the Western blot signals. The band density of HuR protein levels was normalized to the α-tubulin loading control band density and then expressed as a percentage of the HuR levels observed for cells expressing the UBE2D1 Cas9VHH. Figure 12C is a Western blot of U2OS cell lysates after lentiviral transduction of the encoded control (Cas9VHH_UBE2D1) and HuR-binding variant fusion polypeptide constructs, HuR17_UBE2D1 and HuR8_UBE2D1. The Western blot was probed with antibodies against HuR and alpha-tubulin (as a loading control). Figure 12D is a graph showing densitometry of the Western blot signals. The band density of HuR protein levels was normalized to the α-tubulin loading control band density and then expressed as a percentage of the HuR levels observed for cells expressing Cas9VHH_UBE2D1. [Figure 13]Figure 13A is a graph comparing different degradation domains for KRas degradation in HPAC cell lines. HPAC pancreatic cancer cell lines were transduced with lentiviruses encoding PROTACs targeting KRas (using the KRas-binding DARPin K19). PROTACs containing the following degradation domains were investigated: UBE2D1 (E2D1), UBE2B (E2B), and VHL. KRas-targeting PROTACs were tested in both the "binding domain-regulatory domain" and "regulatory domain-binding domain" orientations. The negative control DARPin E3_5 was used as a negative control binding domain in combination with various degradation domains in the following orientation: E3_5-regulatory domain. Figure 13A is a Western blot of KRas and loading control alpha-tubulin expression in cell lysates transduced with lentiviral PROTAC constructs. Figure 13B is a graph of quantification of KRas expression using Western blot densitometry of KRas and alpha-tubulin expression. Data are shown relative to untreated cell-only controls and normalized to α-tubulin loading control levels. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure relates to targeted protein modulation using molecules comprising a targeting moiety and a regulatory domain, and the use of such modulation to study protein function and combat disease. In particular, a first aspect of the disclosure provides a molecule comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugate domain having an amino acid sequence having at least 80%, 85%, 90%, 95%, or 98% sequence identity to a human E2 enzyme or a functional portion thereof; and (b) a targeting domain capable of targeting the regulatory domain to a substrate.

[0031] The term "molecule" includes any entity having a regulatory domain and a targeting domain as defined herein. In preferred embodiments, the molecule is a polypeptide. In some embodiments, the regulatory domain and the targeting domain are linked via a polypeptide linker. Preferably, the molecule is a polypeptide, and the regulatory domain and the targeting domain are linked via a polypeptide linker, as further described herein.

[0032] It will be understood that reference to a regulatory domain and a targeting domain simply refers to the individual portions of the molecule that have the respective regulatory and targeting functions described herein. In this way, the molecules of the present disclosure can be considered to be bifunctional molecules that typically contain two protein-binding domains linked by a linker of appropriate length. Given the different functions of the domains, the molecules will generally be understood to be heterobifunctional.

[0033] By regulatory domain, we mean a portion of a molecule of the present disclosure that can facilitate modulation of a target substrate, such as modulating one or more activities of the target substrate, modulating the cellular location of the target substrate, modulating the stability of the target substrate, and / or modulating the degree of post-translational modification of the target substrate. While a regulatory domain can effect target modulation by any means, it will be understood that because a regulatory domain contains an E2 ubiquitin or ubiquitin-like conjugation domain, modulation is typically mediated by the attachment of a ubiquitin or ubiquitin-like protein to the target substrate. Those skilled in the art will recognize that different ubiquitin or ubiquitin-like proteins conjugated to a target substrate will exert different effects on one or more activities of the target substrate and / or the cellular location and / or stability of the target substrate, depending on which ubiquitin or ubiquitin-like protein is conjugated thereto. Such effects are reviewed in Herrman et al. (Circ Res 2007, 100(9):1276-1291). Conjugation of ubiquitin or ubiquitin-like proteins to target substrates may also modulate the activity of the target substrate through steric effects, such as slowing the rate of a chemical reaction and / or preventing downstream signaling, for example, by steric hindrance. Addition of ubiquitin or ubiquitin-like molecules to a substrate may directly interfere with the interaction of the substrate with a binding partner (e.g., blocking RAS:RAF binding and thereby terminating signaling), due to the size of the ubiquitin / ubiquitin-like protein addition. For the avoidance of doubt, all such effects are encompassed by the term "modulation" as used herein.

[0034] In one embodiment, modulation comprises the target substrate being degraded, or an increase in the stability of the target substrate, or a change in the intracellular location of the target substrate, or one or more activities (e.g., an increase or decrease) of the target substrate being modulated, or the degree of post-translational modification of the target substrate being modulated.

[0035] In certain embodiments, the regulatory domain comprises an E2 ubiquitin conjugation domain that can conjugate ubiquitin to a target substrate, thereby degrading the target substrate. In this manner, it will be understood that when the regulatory domain acts to degrade the target substrate, it can be referred to as a degradation domain.

[0036] In another specific embodiment, the modulation comprises an E2 ubiquitin-like conjugating domain capable of modulating the intracellular location of a target substrate or modulating one or more activities of a target substrate.

[0037] It will thus be appreciated that, depending on the identity of the regulation exerted by the domain, a regulatory domain may be considered to be a degradation domain, a localization domain, an activation domain, or an inactivation domain. In a preferred embodiment, the regulatory domain is a degradation domain.

[0038] In a preferred embodiment, the regulatory domain acts to degrade the target substrate by including an E2 ubiquitin conjugation domain, in which case it may be referred to as a degradation domain.

[0039] Degradation includes the meaning that the amount of the target substrate is reduced by degradation of the target substrate in the proteasome. The amount of the target substrate can be reduced in the presence of a molecule of the present disclosure compared to the amount of the target substrate in the absence of the molecule of the present disclosure. For example, in the presence of a molecule of the present disclosure, the amount of the target substrate can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the amount of the target substrate in the absence of the molecule of the present disclosure. Similarly, when a cell contains a molecule of the present disclosure, it will be understood that the molecule can reduce the amount of the target substrate in the cell by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to the level of the target substrate in the cell in the absence of the molecule of the present disclosure. Preferably, the amount of the target substrate is reduced to an undetectable level. In one embodiment, a molecule of the present disclosure results in degradation of 30-100%, e.g., 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100% of the amount of target substrate that would be present in the absence of the molecule of the present disclosure. It is understood that the substrate may be degraded at background levels, e.g., as part of normal protein turnover, in the absence of the molecule of the present disclosure. In this case, modulation may act to degrade the target substrate at greater than background degradation rates.

[0040] In the case of intracellular target substrates, the extent of degradation can be assessed by measuring the level of the target substrate in cells containing a molecule of the present disclosure and measuring the level of the target substrate in otherwise substantially identical cells that do not contain the molecule of the present disclosure. "Substantially the same" includes the meaning that the cells are of the same type (e.g., express substantially the same cell surface markers), and / or are derived from the same tissue, and / or are in the same stage of the cell cycle. Alternatively, the starting amount of the target substrate in the cells can be measured in the absence of a molecule of the present disclosure, and then the amount of the target substrate can be measured after adding a molecule of the present disclosure to the cells. As yet another alternative, the amount of the target substrate in cells in the presence of a molecule of the present disclosure can be compared to a negative control. "Negative control" includes the meaning of cells in which an inactive form of a molecule of the present disclosure is present, e.g., a molecule that lacks the targeting domain and / or regulatory domain or that includes a non-functional targeting domain and / or regulatory domain. For example, the inactive form may lack a binding domain for the substrate or may have an irrelevant binding domain. Similarly, an inactive form can include an inactive regulatory domain, e.g., one that is unable to interact with one or more binding partners required to mediate regulation. For example, as further described below, including in Example 6, an E2 enzyme variant UBE2D1 containing the mutation F62A completely abolished regulatory activity. Without wishing to be bound by any theory, the inventors believe this is because the F62 residue is involved in the interaction between UBE2D1 and a cyclic E3 ligase such as RNF4. Thus, it will be understood that a negative control can be one in which the E2 protein is unable to interact with an E3 protein, e.g., one that contains a mutation at a position corresponding to F62 in the E2 protein UBE2D1 (e.g., F62A). In another example, an inactive regulatory domain can contain one or more mutations at the catalytic cysteine ​​residue, thereby abolishing its catalytic activity. Exemplary mutations at the catalytic cysteine ​​residues of the regulatory domain include C85A for UBE2D1 and C88A for UBE2B, which abolish regulatory activity. The present disclosure provides examples of such negative control fusion proteins in Table 12A below.Again, it is preferred that the negative control cells are otherwise substantially the same as the cells containing the molecules of the present disclosure. Thus, reference to degradation of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, etc., compared to the amount of substrate in the absence of a molecule of the present disclosure will be understood to include comparison to the amount of substrate in otherwise substantially the same cells that do not contain a molecule of the present disclosure, or comparison to the amount of substrate in cells before addition of a molecule of the present disclosure to the cells, or comparison to the amount of substrate in cells that contain an inactive version of a molecule of the present disclosure.

[0041] Evaluating the level of a target substrate in the presence and absence of a molecule of the present disclosure can be performed using techniques well known in the art. For example, evaluating protein levels is standard practice in the art, and any suitable method can be used. For example, immunoassays such as ELISA or radioimmunoassay, immunofluorescence, HPLC, gel electrophoresis, and capillary electrophoresis (followed by, for example, UV or fluorescence detection) can be used to detect and quantify the target substrate. Methods for measuring the level of a target substrate by mass spectrometry are well known in the art, and any suitable form of mass spectrometry can be used. Western blotting, immunoprecipitation, immunohistochemistry on paraffin, immunofluorescence, fluorescent in situ hybridization, and flow cytometry can also be used. As further described in the Examples, Western blotting and densitometry are convenient methods for detecting and quantifying the target substrate.

[0042] As noted above, in some embodiments, the regulatory domain may be considered a localization domain, an activation domain, or an inactivation domain, depending on which ubiquitin or ubiquitin-like protein becomes bound to the target substrate.

[0043] A localization domain refers to a domain that acts to induce a target substrate to preferentially reside in a specific cellular location (e.g., one or more specific intracellular locations, such as organelles). For example, in the presence of a molecule of the present disclosure, the localization domain of the molecule may result in a higher proportion of the target substrate in a cell residing in a specific intracellular location compared to the proportion of the target substrate remaining in those specific intracellular locations in the absence of the molecule of the present disclosure. Methods for assessing the cellular localization of a target substrate are well known in the art, and any suitable method, such as immunohistochemistry, may be used.Examples of such localization domains and the effect of conjugation of ubiquitin-like proteins to target substrates in the context of localization include Embabe et al. ("Mdm2-mediated NEDDylation of HuR controls the nuclear localization of HuR and protects it from degradation," Hepatology 2012,55(4):1237-48), Wen et al. ("SUMOylation Promotes Nuclear Import and Stabilization of Polo-like Kinase 1 to Support Its Mitotic Function," Cell Rep 2017,21,2147-59), Matunis et al. ("A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex," J Cell Biol 1996,135(6 Pt 1):1457-70), and Mahajan et al. ("A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2,” Cell 1997 88(1):97-107), all of which are incorporated herein by reference.

[0044] By activation domain, we mean a domain that acts to increase one or more activities of a target substrate compared to a reference level of the one or more activities in the absence of a molecule of the present disclosure. The one or more activities may include binding interactions with cellular entities, such as proteins and / or nucleic acids, or enzymatic or signaling activities. The one or more activities may be increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the one or more activities in the absence of a molecule of the present disclosure. Such activities can be assayed using techniques well known in the art, such as ELISA, and one of skill in the art would be able to tailor the assay to the target substrate of interest by consulting the scientific literature. Examples of such activation domains and the effect of conjugation of ubiquitin-like proteins to target substrates in the context of activation include those demonstrated by Soucy et al. (“Cullin-RING ubiquitin E3 ligases require NEDD8 modification to be activated,” Clin Cancer Res 2009,15(12):3912-16) and Noh et al. (“NEDDylation increases RCAN1 binding to calcineurin,” PLoS ONE 2012,7(10):e48315), all of which are incorporated herein by reference.

[0045] By inactivation domain, we include the meaning of a domain that acts to reduce one or more activities of a target substrate compared to a reference level of the one or more activities in the absence of a molecule of the present disclosure. The one or more activities may include binding interactions with cellular entities, such as proteins and / or nucleic acids, or enzymatic or signaling activities. The one or more activities may be reduced by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, preferably to an undetectable level, compared to the one or more activities in the absence of a molecule of the present disclosure. Examples of such inactivation domains and the effect of conjugation of ubiquitin-like proteins to target substrates in relation to inactivation include those demonstrated by Kamynina and Stover (“SREBP SUMOylation inhibits SREBP transcriptional activity indirectly through the recruitment of a co-repressor complex that includes histone deacetylase 3 (HDAC3)” Adv Exp Med Biol 2017, 963:143-68) and Yang et al (“SUMOylation Inhibits SF-1 Activity by Reducing CDK7-Mediated Serine 203 Phosphorylation,” Mol Cell Biol 2009, 29(3):613-25), all of which are incorporated herein by reference.

[0046] The term "E2 ubiquitin or ubiquitin-like conjugating domain" includes reference to a domain capable of binding ubiquitin or a ubiquitin-like protein to a target substrate. For example, the regulatory domain can include an E2 ubiquitin-conjugating domain capable of binding ubiquitin and transferring the ubiquitin to a target substrate. Alternatively, the regulatory domain can include an E2 ubiquitin-like conjugating domain capable of binding a ubiquitin-like protein and transferring the ubiquitin-like protein to a target substrate, such as any one of SUMO, NEDD8, ATG8, ATG12, ISG15, UFM1, FAT10, URM1, and FUBI.

[0047] In some embodiments, the ability of an E2 ubiquitin or ubiquitin-like conjugating domain to bind a target substrate can be assessed when the E2 ubiquitin or ubiquitin-like conjugating domain is part of a molecule of the present disclosure alongside a targeting domain that selectively targets the target substrate in question. Thus, in one embodiment, the E2 ubiquitin or ubiquitin-like conjugating domain in a molecule of the present disclosure is capable of conjugating ubiquitin or a ubiquitin-like protein to a target substrate such that at least 10% of the target substrate is conjugated to the ubiquitin or ubiquitin-like protein. Preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the target substrate is conjugated to the ubiquitin or ubiquitin-like protein. Assessment can be performed in vivo or in vitro. For example, assessment can be performed by recombinant biochemical assay or in a cell.

[0048] It will be understood that conjugation of ubiquitin or ubiquitin-like proteins to target substrates can be assessed directly or indirectly using routine methods in the art. For example, conjugation of ubiquitin or ubiquitin-like proteins to target substrates can be measured directly by detecting a change in the molecular weight of the target substrate as a marker of ubiquitin or ubiquitin-like protein conjugation (e.g., SDS-PAGE separation), or by using Western blots and immunoassays based, for example, on antibodies specific for ubiquitin or ubiquitin-like proteins. Alternatively, conjugation of ubiquitin or ubiquitin-like proteins to target substrates can be measured indirectly, for example, by assessing downstream effects of conjugation, i.e., degradation of the target substrate or another modulation as described herein. Again, any suitable technique can be used for indirect measurement, as is well known in the art and described herein and in the Examples. It will be understood that such assays can be in vivo or in vitro. Illustrative methods for measuring ubiquitin or ubiquitin-like conjugation include cellular assays such as quantitative live-cell assays (see, e.g., Richting et al. (“Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action,” ACS Chem Biol 2018, 13(9):2758-70), biotinylation assays such as in vivo biotinylation assays (see, e.g., Pirone et al. “A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation,” Sci Rep 2017, 7:40756), mass spectrometry, and / or immunostaining. Activity can also be measured using recombinant assays such as those provided by Abcam (Cambridge, UK).

[0049] Humans have approximately 41 E2 enzymes, and the amino acid sequences (and nucleotide sequences of the cDNAs encoding them) are available by reference to GenBank or UniProt. The amino acid and nucleotide sequences encoding various human E2 enzymes are also included in Tables 7-9 below. It will be appreciated that human E2 is compatible with therapeutic use in human cells and is unlikely to elicit an immunogenic response in humans.

[0050] There are various classifications of E2 enzymes. For example, Michelle et al. (J Mol Evol 2009, 68:616-628) classified enzymes into 17 families, families 1 to 17, based on phylogenetic analysis, and all such families are within the scope of the present disclosure. Therefore, the E2 enzymes described herein include any E2 enzyme selected from any one of family 1 E2 enzymes, family 2 E2 enzymes, family 3 E2 enzymes, family 4 E2 enzymes, family 5 E2 enzymes, family 6 E2 enzymes, family 7 E2 enzymes, family 8 E2 enzymes, family 9 E2 enzymes, family 10 E2 enzymes, family 11 E2 enzymes, family 12 E2 enzymes, family 13 E2 enzymes, family 14 E2 enzymes, family 15 E2 enzymes, family 16 E2 enzymes, and family 17 E2 enzymes. Hormaechea-Agulla et al. (Mol Cell 2018, 41(3):168-178) classified enzymes into four classes, classes I to IV. Class I contains only a UBC domain, classes II and III have either N- or C-terminal extensions, respectively, and class IV E2s have both N- and C-terminal extensions. Again, for the avoidance of doubt, all such classes of E2s are included within the scope of this disclosure, and therefore, by E2 enzymes as referred to herein, it is meant class I E2s, class II E2s, class III E2s, and class IV E2s.

[0051] "Having an amino acid sequence having at least 80% sequence identity to a human E2 enzyme or a functional portion thereof" includes the meaning that the E2 ubiquitin or ubiquitin-like conjugate domain must have an amino acid sequence having at least 80% sequence identity to any human E2 enzyme or a functional portion thereof (e.g., any human E2 enzyme listed in Tables 3-9), for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any human E2 enzyme or a functional portion thereof (e.g., any human E2 enzyme listed in Tables 3-9).

[0052] The term "functional portion" includes, for example, as described above, a portion of a human E2 enzyme that has ubiquitin or ubiquitin-like binding ability. Typically, a functional portion is at least 20 amino acids in length, e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. Preferably, a functional portion is 50-150 amino acids in length or 80-150 amino acids in length, e.g., 100-150 amino acids in length. For example, a functional portion of a human E2 enzyme includes a portion of a human E2 enzyme that is capable of conjugating ubiquitin or a ubiquitin-like protein to a target substrate, e.g., when the functional portion is part of a molecule of the present disclosure in conjunction with a targeting domain that selectively targets the target substrate in question. As will become clearer below, the functional portion is preferably a UBC domain, but it will be understood that it may also be a portion of the UBC domain, provided that the portion is still capable of conjugating ubiquitin or a ubiquitin-like protein to a target substrate.

[0053] In one embodiment, the E2 ubiquitin or ubiquitin-like conjugate domain is derived from an E2 enzyme or a functional portion thereof, or is synthetic.

[0054] All E2 enzymes have a core catalytic domain called the UBC domain. Thus, in one embodiment, the E2 ubiquitin or ubiquitin-like conjugation domain comprises the ubiquitin core catalytic (UBC) domain of a human E2 enzyme, or a variant of the UBC domain of a human E2 enzyme that still has ubiquitin or ubiquitin-like conjugation capability, e.g., as described above. Thus, the UBC domain used in the present disclosure may be naturally occurring, e.g., derived from a human E2 enzyme, or synthetic. Synthetic variants may be designed according to consensus sequences within the UBC domain, as described in more detail below.

[0055] The amino acid sequences of the UBC domains of human E2 enzymes are provided below in Table 8. It will be appreciated that the amino acid sequences of the UBC domains of additional E2 enzymes can also be identified by one of skill in the art by searching for a sequence corresponding to one of the UBC domains in Table 8 using standard alignment techniques such as, for example, MacVector and Clustal W. A UBC domain generally consists of four alpha helices and a four-stranded beta sheet.

[0056] The length of UBC in human E2 enzymes ranges from 117 to 284 amino acids, and therefore, in one embodiment, the UBC domain comprises 110 to 290 amino acids, for example, 117 to 284 amino acids or 140 to 192 amino acids.

[0057] Comparison of UBC domains has identified important conserved regions or consensus sequences therein, as described, for example, by Michelle et al. ("What was the set of ubiquitin and ubiquitin-like conjugating enzymes in the eukaryotic common ancestor?" J Mol Evol 2009, 68:616-628; see particularly Figure 6 therein). For example, a common signature motif is the HxN tripeptide (e.g., HPN or histidine proline asparagine) with an active cysteine ​​residue typically located eight amino acids C-terminal to this canonical motif. The PxxxP (SEQ ID NO: 206) motif and the tryptophan residue 26-43 amino acids C-terminal to the PxxxP motif (SEQ ID NO: 206) are also conserved.

[0058] Thus, in one embodiment, the E2 ubiquitin or ubiquitin-like conjugating domain comprises a UBC domain containing a conserved catalytic cysteine ​​residue. However, it will be understood that a UBC domain does not necessarily require a catalytic cysteine ​​residue. For example, UBE2V1 and UBE2V2 lack the conserved cysteine ​​residue but still interact with Ube2N to enable lysine 63 (K63) polyubiquitin chain formation. In other words, it will be understood that a UBC domain may become active in the cellular environment, for example, through interactions with other E2 proteins. Nevertheless, it is preferred that the E2 ubiquitin or ubiquitin-like conjugating domain is catalytic and has the conserved cysteine ​​residue.

[0059] In another embodiment, the E2 ubiquitin or ubiquitin-like conjugate domain comprises a UBC domain that includes an HxN peptide motif, such as an HPN tripeptide. Thus, it will be understood that a UBC domain can include the HxN peptide motif (HPN tripeptide) and the conserved cysteine ​​residue generally located at the eighth amino acid C-terminal to this canonical motif.

[0060] Using the characterization of E2s into 17 families described by Michelle et al. (J Mol Evol 2009, 68:616-628), family 5 (the human E2s in this family are UBE2J1 and UBE2J2) missed the canonical tripeptide HxN, which was replaced by TPNGRF (SEQ ID NO:208) or TANGRF (SEQ ID NO:209). Thus, in a further embodiment, the UBC domain may contain a TxNGRF (SEQ ID NO:210) peptide motif, e.g., TPNGRF (SEQ ID NO:208) or TANGRF (SEQ ID NO:209), instead of the HxN motif. Thus, the UBC may contain a TxNGRF (SEQ ID NO:210) peptide motif, e.g., TPNGRF (SEQ ID NO:208) or TANGRF (SEQ ID NO:209), and a conserved cysteine ​​residue.

[0061] In a further embodiment, the E2 ubiquitin or ubiquitin-like conjugate domain comprises a UBC domain that includes a PxxxP (SEQ ID NO: 206) peptide motif, such as a PxxPP (SEQ ID NO: 207) motif.

[0062] In a further embodiment, the E2 ubiquitin or ubiquitin-like conjugating domain comprises a UBC domain containing a conserved tryptophan residue, preferably between 26 and 43 amino acids from the C-terminus of a PxxxP motif (SEQ ID NO: 206), e.g., a PxxPP (SEQ ID NO: 207) motif.

[0063] In still further embodiments, the E2 ubiquitin or ubiquitin-like conjugate domain comprises (i) a conserved cysteine ​​residue; and / or (ii) an HxN peptide motif, e.g., HPN, or a TxNGRF (SEQ ID NO: 210) peptide motif, e.g., TPNGRF (SEQ ID NO: 208) or TANGRF (SEQ ID NO: 209); and / or (iii) a PxxxP (SEQ ID NO: 206) peptide motif, e.g., a PxxPP (SEQ ID NO: 207) motif; and / or (iv) a UBC domain containing a conserved tryptophan residue.

[0064] In still further embodiments, the E2 ubiquitin or ubiquitin-like conjugate domain comprises a UBC domain comprising: (i) a conserved cysteine ​​residue; (ii) an HxN peptide motif, e.g., HPN, or a TxNGRF (SEQ ID NO: 210) peptide motif, e.g., TPNGRF (SEQ ID NO: 208) or TANGRF (SEQ ID NO: 209); (iii) a PxxxP (SEQ ID NO: 206) peptide motif, e.g., a PxxPP (SEQ ID NO: 207) motif; and (iv) a conserved tryptophan residue, wherein the conserved tryptophan residue is 26-34 amino acids from the C-terminus of the PxxxP motif (SEQ ID NO: 2206), and the conserved cysteine ​​residue is within 8 amino acids of the C-terminus of the HxN or TxNGRF motif.

[0065] In one embodiment, the ubiquitin or ubiquitin-like conjugate domain comprises a UBC domain that is a variant of the UBC of a human E2 enzyme, where the variant shares at least 80% sequence identity with the UBC of the human E2 enzyme. For example, variants include UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UbcH1C), UBE2G2 (UbcH1D), UBE2G3 (UbcH1D), UBE2G4 (UbcH1D), UBE2G5 (UbcH5C), UBE2G6 (UbcH1D), UBE2G7 (UbcH1D), UBE2G8 (UbcH1D), UBE2G9 (UbcH1D), UBE2G1 (UbcH1D), UBE2G1 (UbcH1D), UBE2G2 (UbcH1D), UBE2G3 (UbcH1D), UBE2G4 (UbcH1D), UBE2G5 (UbcH1D), UBE2G6 (UbcH1D), UBE2G7 (UbcH1D), UBE2G8 (UbcH1D), UBE2G9 (UbcH1D), UBE2G1 (UbcH1D), UBE2G1 (UbcH1D), UBE2G1 (UbcH1D), UBE2G2 (UbcH1D), UBE2G3 (UbcH1D), UBE2G4 (UbcH1D), UBE2G5 (UbcH1D), UBE2G6 (UbcH1D), UBE2 BE2G), UBE2G2(UBC7), UBE2H(UBCH), UBE2I(Ubc9), UBE2J1(NCUBE1), UBE2J2(NCUBE2), UBE2K(HIP2 ), UBE2L3 (UbcH7), UBE2L6 (UbcH8), UBE2M (Ubc12), UBE2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q 1(NICE-5), UBE2Q2, UBE2QL, UBE2R1(CDC34), UBE2R2(CDC34B), UBE2S(E2-EPF), UBE2T(HSPC150), U BE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (A It may have an amino acid sequence that is at least 80% sequence identity (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the UBC domain of any one of KTIP, TSG101, and UFC1 (SEQ ID NOs: 42 to 82, respectively).

[0066] The percent sequence identity between two polypeptides can be determined using any suitable computer program, such as the GAP program of the University of Wisconsin Genetic Computing Group, with it being understood that the percent identity is calculated for polypeptides whose sequences are optimally aligned. Alignment may alternatively be performed using the Clustal W program (Thompson et al. Nucleic Acids Res 1994, 22(22):4673-80). Parameters used may be as follows: Fast pairwise alignment parameters: K tuple (word) size; 1, window size; 5, gap penalty; 3, number of upper diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Score matrix: BLOSUM.

[0067] Typically, such variants that share at least 80% sequence identity with the UBC of a human E2 enzyme still retain the conserved sequence of the UBC domain, as described above. Thus, variants of the UBC of a human E2 enzyme preferably include (i) a conserved cysteine ​​residue; and / or (ii) an HxN peptide motif, such as HPN, or a TxNGRF (SEQ ID NO: 210) peptide motif, such as TPNGRF (SEQ ID NO: 208) or TANGRF (SEQ ID NO: 209); and / or (iii) a PxxxP (SEQ ID NO: 206) peptide motif, such as a PxxPP (SEQ ID NO: 207) motif; and / or (iv) a conserved tryptophan residue. Most preferably, the variant of the UBC of the human E2 enzyme comprises: (i) a conserved cysteine ​​residue; (ii) an HxN peptide motif, e.g., HPN, or a TxNGRF (SEQ ID NO: 210) peptide motif, e.g., TPNGRF (SEQ ID NO: 208) or TANGRF (SEQ ID NO: 209); (iii) a PxxxP (SEQ ID NO: 206) peptide motif, e.g., a PxxPP (SEQ ID NO: 207) motif; and (iv) a conserved tryptophan residue, wherein the conserved tryptophan residue is 26 to 34 amino acids from the C-terminus of the PxxxP motif (SEQ ID NO: 206) and the conserved cysteine ​​residue is within 8 amino acids of the C-terminus of the HxN or TxNGRF motif (SEQ ID NO: 210).

[0068] Variants include variants of the UBC domain whose amino acid sequence comprises one or more deletions and / or one or more amino acid substitutions; and / or one or more insertions compared to the amino acid sequence of the parent human E2 enzyme UBC.

[0069] Variants may be produced by any suitable method: traditional site-directed mutagenesis may be used, or polymerase chain reaction-based procedures well known in the art may be used.

[0070] Typically, the amino acid substitutions in the variants disclosed herein are preferably conservative amino acid substitutions, for example, where an amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative amino acid substitutions are well known in the art and include (original residue <-> substitution): Ala (A) <-> Val, Gly, or Pro; Arg (R) <-> Lys or His; Asn (N) <-> Gln; Asp (D) <-> Glu; Cys (C) <-> Ser; Gln (Q) <-> Asn; Glu (G) <-> Asp; Gly (G) <-> Ala; His (H) <-> Arg; Ile ( Includes I)<->Leu; Leu(L)<->Ile, Val, or Met; Lys(K)<->Arg; Met(M)<->Leu; Phe(F)<->Tyr; Pro(P)<->Ala; Ser(S)<->Thr or Cys; Thr(T)<->Ser; Trp(W)<->Tyr; Tyr(Y)<->Phe or Trp; and Val(V)<->Leu or Ala.

[0071] In a preferred embodiment, the ubiquitin or ubiquitin-like conjugate domain is a UBC domain of a human E2 enzyme, such as UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UBE2G), UBE2G2 (UBC7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (Ub cH7), UBE2L6 (UbcH8), UBE2M (Ubc12), UBE2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1, and the amino acid sequences of the UBC domains are identified by SEQ ID NOs: 42 to 82, respectively.

[0072] It will be appreciated that BIRC6 and UBE2O are described in the art as E2 / E3 hybrid enzymes, as they are E3-independent E2 ubiquitin-conjugating enzymes (see Bartke et al. Mol Cell 2004 and Ullah et al. FEBS J 2018). For the avoidance of doubt, such enzymes are included in the definition of E2 enzymes herein.

[0073] To avoid any misunderstanding by "human E2" herein, we include the meaning of "derived from human E2" such that although the cDNA or gene expressing the enzyme was originally obtained using genetic material of human origin, the protein can then be expressed in any host cell. Thus, it will be clear that human E2 can be expressed in a prokaryotic host cell, such as E. coli, and still be considered to be human E2.

[0074] In a further embodiment, the regulatory domain comprises an E2 enzyme, wherein the E2 enzyme comprises an E2 ubiquitin or ubiquitin-like conjugate domain. It will thus be understood that the regulatory domain can comprise the full-length E2 enzyme, not just its UBC domain or another functional portion thereof.

[0075] When the regulatory domain comprises an E2 enzyme, the E2 enzyme has an amino acid sequence having at least 80% sequence identity to any human E2 enzyme, such as those listed in Tables 3-8 below, for example, those having an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human E2 enzyme. Preferably, the E2 enzyme is UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2E4 (UbcH10), UBE2E5 (UbcH15), UBE2E6 (UbcH16), UBE2E7 (UbcH17), UBE2E8 (UbcH19), UBE2E9 (UbcH18), UBE2E10 (UbcH19), UBE2E11 (UbcH15), UBE2E12 (UbcH15), UBE2E13 (UbcH16), UBE2E14 (UbcH18), UBE2E1 BE2F(NCE2), UBE2G1(UBE2G), UBE2G2(UBC7), UBE2H(UBCH), UBE2I(Ubc9), UBE2J1(NCUBE 1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (UbcH7), UBE2L6 (UbcH8), UBE2M (Ubc12), UBE2 and UFC1, and the amino acid sequences thereof are provided in SEQ ID NOs: 1 to 41, respectively (see Table 7). Most preferably, the E2 enzyme is UBE2D1 (UbcH5A), UBE2E2, UBE2L3 (UbcH7), UBE2O (E2-230K), UBE2Q2, or UBE2R2.

[0076] Thus, it will be understood that the E2 enzyme can be a variant of any of the human E2 enzymes described herein (see, e.g., Tables 3-9) that has at least 80% sequence identity with any one of the human E2 enzymes, e.g., as provided in SEQ ID NOS: 1-41. Variant includes the meaning of the amino acid sequence of a human E2 enzyme that includes one or more deletions and / or one or more amino acid substitutions; and / or one or more insertions. Amino acid substitutions are preferred, including the conservative amino acid substitutions described above. Thus, it will be understood that the regulatory domain can include a human E2 enzyme or the UBC domain of a human E2 enzyme (e.g., any of those described herein, e.g., listed in Tables 3-9) in which up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or up to 30 amino acids have been added, deleted, and / or substituted (e.g., conservatively substituted) by other amino acids. Generally, mutations are limited to cysteine ​​residues important for catalytic activity, HxN motifs, PxxxP motifs (SEQ ID NO: 206), and other conserved regions described herein, including cysteine ​​residues. Similarly, mutations generally do not disrupt interactions between the E2 enzyme and one or more binding partners involved in mediating the regulatory function of the E2 enzyme. For example, as described in Example 6, the E2 enzyme variant UBE2D1 containing the F62A mutation completely abolished the regulatory activity that was thought to be due to the involvement of the F62 residue in the interaction between UBE2D1 and endogenous RING-type E3 ligases, such as RNF4. Thus, mutant forms of E2 enzymes typically remain capable of interacting with E3 enzymes (e.g., E3 proteins that naturally bind to perform the desired regulatory function). "Still able to interact with E3 enzyme" includes the meaning that the variant of the E2 enzyme exhibits at least 50% of the binding to the E3 enzyme, for example 60%, 70%, 80% or 90% of the binding, more preferably 95% or 99% of the binding to the E3 enzyme, as an unaltered level of binding between the E2 enzyme and the E3 enzyme.Methods for assessing protein-protein interactions are standard practice in the art, including E2:E3 binding pairs (e.g., Gundogdu and Walden, Protein Science. 2019;28:1758-1770; Ning Zheng and Nitzan Shabek. Annual Rev Biochemistry, Vol. 86:129-157, 2017; and Turek et al., JBC 293, 16324-16336, 2018).

[0077] Additionally, to minimize self-ubiquitination of the molecules of the present disclosure, it may be desirable to modify the human E2 enzyme or a functional portion thereof (e.g., the UBC domain), for example, by modifying any one or more lysine residues within the human E2 enzyme or a functional portion thereof. By "modification" we mean one or more amino acid substitutions (e.g., conservative substitutions), deletions, and / or additions. This can be accomplished using standard recombinant techniques, such as conventional site-directed mutagenesis, or by PCR. Such modified human E2 enzymes may also be considered variants. Such modifications (e.g., where one or more lysine residues are replaced with another amino acid) may also increase the stability of the resulting protein, for example, if the modifications are stabilizing modifications, e.g., based on modeling predictions from protein crystal structures.

[0078] Additionally or alternatively, to enhance the stability of the molecules of the present disclosure, it may be desirable to modify the human E2 enzyme or a functional portion thereof (e.g., the UBC domain), e.g., by modifying any one or more amino acid residues within the human E2 enzyme or a functional portion thereof that are known to be stabilizing, e.g., based on modeling predictions from protein crystal structures. Again, "modified" includes one or more amino acid substitutions (e.g., conservative substitutions), and / or deletions and / or additions. Such modified human E2 enzymes may also be considered variants.

[0079] It will thus be understood that a regulatory domain can comprise a variant of one of the amino acid sequences of any one of SEQ ID NOs: 1-82 (i.e., any of the human E2 enzymes or UBC domains thereof in Tables 3-9) comprising up to 30 amino acid modifications, for example, 1, or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or up to 15, 20, 25, or up to 30 amino acid modifications. Modifications can, for example, minimize autoubiquitination and / or increase stability.

[0080] In a preferred embodiment, the regulatory domain is selected from the group consisting of UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UBE2G), UBE2G2 (UBC7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (UbcH7), UBE2L6 (UbcH8), The E2 enzymes include UBE2M (Ubc12), UBE2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1 (the E2 enzymes are selected from the group consisting of the amino acid sequences identified by SEQ ID NOs: 1 to 41, respectively).

[0081] It will be understood that any of the examples of possible regulatory domains described above and listed in SEQ ID NOS: 1-82 may contain up to five amino acids (e.g., up to two amino acids) at one or both termini, which may result from the cloning strategy employed to express them. However, it will be understood that these amino acids should not alter the function of the regulatory domain. For example, the regulatory domain of the von Hippel Lindau (VHL) protein in SEQ ID NOS: 147 contains two amino acids, alanine and methionine, at the N-terminus resulting from the cloning strategy, whereas in SEQ ID NOS: 199, the VHL regulatory domain lacks these two amino acids. However, in both cases, the regulatory domain has the relevant functionality.

[0082] By targeting domain, we include the meaning of any domain or moiety that is capable of targeting a target substrate. Preferably, the targeting domain is capable of selectively targeting the substrate. For example, it is preferred if the targeting domain targets the substrate to a greater extent than any other substrate, and preferably targets only the substrate.

[0083] In one embodiment, the targeting domain binds to a substrate, preferably specifically binds to the substrate. For example, it is preferred that the targeting domain binds to the substrate to a greater extent than any other substrate in the cell in which the molecule of the present disclosure is intended to be used (e.g., the cell containing the substrate to be regulated). For example, it is preferred that the targeting domain has a Kd value (dissociation constant) that is at least 5 or 10 times lower (i.e., higher affinity) than at least one other substrate in the cell, preferably more than 100 or 500 times lower. More preferably, the targeting domain of the substrate has a Kd value that is 1,000 or 5,000 times lower than at least one other substrate in the cell. The Kd value can be easily determined using methods well known in the art.

[0084] The targeting domain is typically a polypeptide (e.g., one that selectively binds to a substrate), such as any one of a monobody, nanobody, antibody, antibody fragment, scFv, intrabody, minibody, scaffold protein, such as a designed ankyrin repeat protein (DARPin), peptide binder or ligand binding domain, preferably one that binds specifically (e.g., a ligand binding domain that can bind to a target substrate with high (e.g., nanomolar Kd or better) affinity).

[0085] As used herein, the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, fragments produced by a Fab expression library, and bispecific antibodies. Such fragments include fragments of whole antibodies that retain their binding activity to a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single-chain antibodies (scFv), fusion proteins, and other synthetic proteins containing the antigen-binding site of an antibody. Targeting domains containing only a portion of an antibody may be advantageous by optimizing clearance rate from the blood and may be less susceptible to nonspecific binding due to the Fc portion. Also included are domain antibodies (dAbs), diabodies, camelid antibodies, and engineered camelid antibodies. Furthermore, for administration to humans, antibodies and fragments thereof can be humanized antibodies, which are now well known in the art (Janeway et al., 2001, Immunobiology., 5th ed., Garland Publishing; An et al., 2009, Therapeutic Monoclonal Antibodies: From Bench to Clinic, ISBN: 978-0-470-11791-0).

[0086] Monobodies, nanobodies, intrabodies, unibodies, asymmetric IgG-like antibodies (e.g., TrionPharma / FreseniusBiotech; knobs-into-holes, Genentech; CrossMAbs, Roche; electrostatically matched antibodies, AMGEN; LUZ-Y, Genentech; strand exchange engineered domain (SEED) bodies, EMD Serono; Bioron, Merus; Fab-exchange antibodies, Genmab), symmetric IgG-like antibodies (e.g., dual targeting (DT)-Ig, GSK / Domantis; two-in-one antibodies, Genentech; cross-linked MAb, Karmanos Cancer Center; mAb2, F-star; and CovX-body, CovX / Pfizer), IgG fusions (e.g., dual variable domain (DVD)-Ig, Abbott; IgG-like bispecific antibodies, EliLilly; Ts2Ab, Mediimmune / AZ; BsAb, ZymoGenetics; HERCULES, BiogenIdec; TvAb, Roche), Fc fusions (e.g., ScFv / Fc fusions, Academic Institution; SCORPION, EmergentBioSolutions / Trubion, ZymoGenetics / BMS; dual affinity retargeting gtechnology (Fc-DART), MacroGenics; bivalent (ScFv)2-Fab, National Research Center for Antibody Medicine), Fab fusions (e.g., F(ab)2, Medarex / AMGEN; dual-action or Bis-Fab, Genentech; Dock-and-Lock (DNL), ImmunoMedics; bivalent bispecific, Biotechnol; and Fab-Fv, UCB-Celltech), ScFv- and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs), Micromet; tandem diabodies (Tandab), Affimed; DARTs, MacroGenics; single-chain diabodies, Academic; TCR-like antibodies, AIT, ReceptorLogics; human serum albumin ScFv fusions, Merrimack;and COMBODIES, EpigenBiotech), IgG / non-IgG fusions (e.g., immunocytokins, EMDSerono, Philogen, ImmunGene, ImmunoMedics; superantibody fusion proteins, ActiveBiotech; and immunomobilizing mTCRAgainstCancer, ImmTAC) and oligoclonal antibodies (e.g., Symphogen and Merus);

[0087] Antibodies are incorporated herein with their specificity-determining regions as described herein by Carter (see "Potent antibody therapeutics by design", Nat Rev Immunol 2006, 6(5):343-57, and Carter ("Introduction to current and future protein therapeutics: a protein engineering perspective", Exp Cell Res 2011, 317(9):1261-9). Thus, the term "antibody" also includes affibodies and non-immunoglobulin frameworks. Examples include adnectins, anticalins, affilins, transbodies, DARPins, Tn3 molecules, trimer X, microproteins, fynomers, avimers, sengulins, and carbitol (ecallantide).

[0088] A targeting domain suitable for a given target substrate can be generated by one of skill in the art using techniques long established in the art. For example, methods for preparing monoclonal antibodies and antibody fragments are well known in the art and include hybridoma technology (Kohler & Milstein, "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature 1975, 256:495-497); antibody phage display (Winter et al., "Making antibodies by phage display technology" Annu Rev Immunol 1994, 12:433-455); ribosome display (Schaffitzel et al., "Ribosome display: an in vitro method for selection and evolution of antibodies from libraries" J Immunol Methods 1999, 231:119-135); and iterative colony filter screening (Giovannoni et al., "Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening" Nucleic Acids Res 2001, 29:E27). Additionally, antibodies and antibody fragments suitable for use in the present disclosure are described, for example, in the following publications:“Monoclonal Hybridoma Antibodies: Techniques and Application”, Hurrell(CRC Press, 1982); “Monoclonal Antibodies: A Manual of Techniques”, H. Zola, CRC Press, 1987, ISBN: 0-84936-476-0; York,1988.ISBN 0-87969-314-2;“Using Antibodies:A Laboratory Manual” 2nd Edition,Harlow & Lane,Eds,Cold Spring Harbor Laboratory Press,New York,1999.ISBN 0-87969-543-9;and“Handbook of Therapeutic Antibodies”Stefan Duebel,Ed.,1st Edition,- Wiley-VCH, Weinheim, 2007.ISBN:3-527-31453-9.

[0089] The targeting domains of the present disclosure can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific targeting domains can be specific for different epitopes of a substrate, or can be specific for both a substrate polypeptide of the present disclosure and a heterologous composition, such as a heterologous polypeptide or solid support material. It will be appreciated that such multispecific targeting domains can be valuable for targeting more complex multidomain substrates.

[0090] In order to minimize the ubiquitination of the targeting domain of the molecule of the present disclosure, it may be desirable to minimize the number of lysine residues in targeting domain.Therefore, targeting domain can be modified by substituting lysine amino acid with, for example, arginine residue.The technique for doing so is well known in the art.

[0091] Specific examples of suitable targeting domains are illustrated in the Examples and include the monobody aCS3, which selectively binds to the C-SH2 domain of Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2); the nanobodies HuR8 and HuR17, which bind to human antigen R; the DARPin K19, which binds to the KRas protein; and Cas9, which selectively binds to the bacterial Cas9 protein (as it is not expressed in mammals), which is utilized herein as an example of a negative control. The amino acid sequences of these targeting domains, as well as a lysine variant of aCS3, are included in Table 10, and it will be understood that any such targeting domain can be used in the context of the present disclosure. Thus, in one embodiment, the targeting domain has the amino acid sequence of any one of SEQ ID NOs: 126-135, 138-139, or 257, or a variant thereof having up to 20 amino acid modifications, e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, 15, or 20 amino acid modifications. By "modified" is meant one or more amino acid substitutions (e.g., conservative substitutions), and / or additions and / or deletions. In another embodiment, the targeting domain has the amino acid sequence of any one of SEQ ID NOs: 126-135, 138-139, 257, or a variant thereof having at least 80% sequence identity to any one of SEQ ID NOs: 126-135, 138-139, 257, e.g., at least 85% or 90% sequence identity to any one of SEQ ID NOs: 126-135, 138-139, 257, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. It will be understood that a variant of the targeting domain can be one that has been modified to minimize ubiquitination of the targeting domain, for example, by modifying one or more lysine residues (e.g., by substituting one or more of them with another amino acid residue and / or deleting one or more of them), and / or to increase the stability of the targeting domain by making one or more modifications that are known to increase stability, for example, based on modeling predictions from protein crystal structures.

[0092] In some embodiments, the present disclosure provides a variant of the amino acid sequence of any one of SEQ ID NOs: 126-135, 138-139, 257, in which one or more of the lysine residues have been substituted with another amino acid and / or deleted; and / or the regulatory domain is a variant of the amino acid sequence of any one of SEQ ID NOs: 42-82, in which one or more of the lysine residues have been substituted with another amino acid and / or deleted. Provide the molecule.

[0093] Substrate (or target substrate) includes the meaning of any substrate that can be targeted by a molecule of the present disclosure, thereby conjugated to ubiquitin or a ubiquitin-like protein, and thereby regulated (e.g., degraded).

[0094] Preferably, the target substrate is a polypeptide, typically an intracellular polypeptide, by which we mean any polypeptide having at least one portion that is intracellular. Thus, the substrate may be an intracellular polypeptide present in the cytosol and / or an organelle within the cell, or may be a membrane polypeptide, such as a transmembrane polypeptide (e.g., GPCR) having at least an intracellular portion. However, ubiquitin is found in both intracellular and extracellular fluids and is involved in the regulation of numerous cellular processes. Extracellular ubiquitin has been implicated in the modulation of immune responses (Sujashvili, "Advantages of extracellular ubiquitin in modulation of immune responses," Mediators Inflamm 2016, Epub 2016:4190390); and Baska et al. suggest that components of the ubiquitin-proteasome pathway are secreted into the epididymal fluid (EF) of mammals (Baska et al., "Mechanism of extracellular ubiquitination in the mammalian epididymis," J Cell Physiol 2008, 215(3):684-96). Thus, it will be appreciated that in certain circumstances, the molecules of the present disclosure may be used to modulate extracellular target substrates. Preferably, however, the substrate is an intracellular polypeptide.

[0095] In one embodiment, the substrate is localized to one or more of the plasma membrane, cytoplasm, nucleus, mitochondria, endosomes, endoplasmic reticulum, mitochondria, and Golgi apparatus.

[0096] Examples of possible target substrates include oncogenic proteins, signaling proteins, GPCRs, post-translationally modified proteins, adhesion proteins, receptors, cell cycle proteins, checkpoint proteins, viral proteins, prion proteins, bacterial proteins, parasitic proteins, fungal proteins, DNA-binding proteins, structural proteins, enzymes, immunogens, antigens, and / or pathogenic proteins. It will be understood that the target substrate may be any potential therapeutic target, whether conventionally druggable or not currently druggable.

[0097] In certain embodiments, the substrate is selected from the group consisting of Ras, KRas, and SHP2. Other possible target substrates include human rhinovirus (HRV) protease 3C, muscarinic acetylcholine receptor 2 (M2R), beta-2 adrenergic receptor (β2-AR), cross-binding endonuclease MUS81 (MUS81), and human antigen R (HuR).

[0098] In some embodiments, the regulatory domain and the targeting domain are connected by a linker. By linker, we mean a chemical moiety that connects the regulatory domain to the targeting domain. It is preferred if the regulatory domain is covalently linked to the targeting domain, for example, by a linker.

[0099] Thus, the regulatory and targeting domains can be linked by any conventional method of linking molecules, such as those generally described by O'Sullivan et al. ("Comparison of two methods of preparing enzyme-antibody conjugates: Application of these conjugates for enzyme immunoassay," Anal Biochem 1979, 100:100-8). For example, one of the regulatory or targeting domains can be enriched with thiol groups, and the other can be reacted with a bifunctional agent capable of reacting with those thiol groups, such as N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), a heterobifunctional crosslinker that incorporates a disulfide bridge between the conjugated species. Amide and thioether bonds, such as those achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulfide bonds. Bismaleimide reagents are known to allow the conjugation of a thiol group (e.g., the thiol group of a cysteine ​​residue of an antibody) to another thiol-containing moiety (e.g., the thiol group of a T cell antigen or a linker intermediate) in a sequential or simultaneous manner. Other functional groups besides maleimides that are reactive with thiol groups include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate.

[0100] In particularly preferred embodiments, the regulatory domain and the targeting domain are polypeptides, and the regulatory domain is linked to the targeting domain directly, without a linker, or indirectly, via a linker.

[0101] It will be understood that the regulatory domain and the targeting domain can be components of a fusion polypeptide that can be encoded by a nucleic acid molecule. Thus, in a particularly preferred embodiment, the molecule of the present disclosure is a fusion polypeptide comprising (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to the human E2 enzyme or a functional portion thereof, and (b) a targeting domain capable of targeting the regulatory domain to a substrate. The regulatory domain can be N-terminal to the targeting domain, or the targeting domain can be N-terminal to the regulatory domain. Fusion polypeptide refers to a protein or polypeptide having amino acid sequences derived from two or more proteins, such as the two heterologous domains described above, i.e., the regulatory domain and the targeting domain. Fusion proteins can also include linking a region of amino acids between amino acid moieties derived from separate proteins.

[0102] Suitably, the regulatory domain and the targeting domain are linked such that both domains retain their respective activities, thereby enabling the molecule to be targeted to a target substrate and subsequently modulating the substrate. Therefore, it may be desirable for the fusion polypeptide to include a peptide linker between the regulatory domain and the targeting domain, for example, to prevent steric disruption between the targeting substrate and the target substrate. Suitable linker peptides typically adopt a random coil conformation, and therefore the linker may contain glycine, serine, or a mixture of glycine and serine residues. Other amino acids that the linker may contain include any one or more of leucine, glutamate, arginine, proline, alanine, asparagine, tyrosine, aspartate, valine, and threonine. Preferably, the linker contains 1 to 45 amino acid residues, e.g., 5 to 28 amino acid residues in length, more preferably 1 to 20 amino acid residues, or 4 to 20 amino acid residues, e.g., 5 to 19 amino acid residues in length. Most preferably, the linker comprises a length of 6 to 20 amino acid residues, for example 9 to 19. Particular lengths of linkers include lengths of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acid residues.

[0103] Examples of particularly preferred linkers that may be used are listed in Table 10 and include the peptides GGGGS (SEQ ID NO: 146) or GGGGGSGGGGSGGGGS (SEQ ID NO: 145) or LEGGGGSSR (SEQ ID NO: 141) or LEGGGGSGGGGSGGGGSSR (SEQ ID NO: 142) AAAGGGGSGGGGSGGGGSGT (SEQ ID NO: 143) or GGGGG (SEQ ID NO: 144) or LEGGSR (SEQ ID NO: 211) or LEGGGSSGGSSR (SEQ ID NO: 212) or LEGGGGSGGGSSR (SEQ ID NO: 213) or LEGGGSGGGSGGGSSR (SEQ ID NO: 214) or LEGGGGSGPSGGGGPS GSR (SEQ ID NO: 215) or LESNGGGGSPAPAPGGGGSGSSR (SEQ ID NO: 216) or LEGGGGSYPYDVPDYASGGGGSSR (SEQ ID NO: 217) or TGGSAGGSGGSAGGSGGSAGGSGGSA (SEQ ID NO: 218) or AGSGGSTGSGGSPTPSTSGGSTGSGGAS (SEQ ID NO: 219) or AGSGGSGGSGGSGNSSTSGGSGGSGGAS (SEQ ID NO: 220) or GGSPVPSTPGGGSGGGSGGSPVPSTPGS (SEQ ID NO: 221) or SPGTGSPGTGSPGTGSPGTGSPGTGSPG (SEQ ID NO: 222). It will be understood that the "LE" and "SR" in these examples are present due to restriction cloning sites introduced into the nucleotide sequence. It will further be understood that one or more serine residues in any of these exemplary linkers can be substituted with a glycine residue.

[0104] Polynucleotides encoding suitable targeting domains are known in the art or can be readily designed from known sequences, for example, from sequences of proteins known to interact with target substrates or from those contained in nucleotide sequence databases such as the GenBank, EMBL, and dbEST databases. Polynucleotides encoding suitable regulatory domains are known in the art or can be readily designed and made from known E2 enzyme sequences.

[0105] A polynucleotide encoding a suitable linker peptide can be readily designed and prepared from the linker peptide sequence.

[0106] Thus, polynucleotides encoding the fusion polypeptides of the present disclosure can be readily constructed using well-known genetic engineering techniques.

[0107] The nucleic acid is then expressed in a suitable host to produce a molecule of the present disclosure, e.g., a fusion polypeptide. Thus, a nucleic acid encoding a fusion polypeptide of the present disclosure can be used in accordance with known techniques, appropriately modified in light of the teachings contained herein, to construct expression vectors that are used to transform suitable host cells for the expression and production of the fusion polypeptides of the present disclosure.

[0108] It will be understood that the nucleic acids encoding the polypeptides of the present disclosure may be joined to a wide variety of other nucleic acid sequences for introduction into an appropriate host. The companion nucleic acid will depend on the nature of the host, the manner of the introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired, as is well known in the art.

[0109] As discussed above and demonstrated in the Examples, the inventors have discovered that it is possible to provide targeted regulation of a target substrate by a molecule comprising a regulatory domain that contains an E2 ubiquitin or ubiquitin-like conjugating domain, rather than an E3 ligase. Thus, in one embodiment, the molecule or fusion polypeptide of the present disclosure does not comprise an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof. By functional portion of an E3 ubiquitin or ubiquitin-like ligase, the inventors include a portion of an E3 ubiquitin or ubiquitin-like ligase that is still capable of assisting the transfer of ubiquitin or ubiquitin-like proteins to a substrate, e.g., directly (as with a HECT E3 ubiquitin ligase) or indirectly (as with a RING E3 ubiquitin ligase). Assaying E3 ubiquitin or ubiquitin-like ligase activity can be performed using any suitable technique known in the art and can include testing whether the E3 ubiquitin or ubiquitin-like ligase can bind to a substrate and an E2-Ub or E2-Ubl (see, e.g., the ternary complex formation assay described by Richting et al. ("Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action," ACS Chem Biol 2018, 13(9):2758-70)). "Not containing an E3 ubiquitin or ubiquitin-like ligase" includes the meaning that the molecule or polypeptide of the disclosure is not covalently linked to an E3 ubiquitin or ubiquitin-like ligase. For example, when the molecule of the disclosure is a fusion polypeptide, the nucleotide sequence encoding the fusion polypeptide also does not encode an E3 ubiquitin or ubiquitin-like ligase.

[0110] In one embodiment, a molecule of the present disclosure (e.g., a polypeptide of the present disclosure) does not comprise an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof, and the E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof comprises one or more domains selected from the group consisting of a RING (Really Interesting New Gene) domain, a U-box domain, a HECT (homologous to the E6-AP carboxyl terminus) domain, and an RBR domain. In one embodiment, a molecule of the present disclosure comprises a subcellular localization signal, such as a nuclear localization signal, a mitochondrial localization signal, or an endosomal localization signal.

[0111] Examples of fusion polypeptides of the present disclosure include those listed in Table 12A, and therefore in preferred embodiments, a molecule of the present disclosure is any one of the fusion polypeptides listed in Table 12A having SEQ ID NOs: 156-167, 170-195, 202-205, 236-248, 253-256, and 266-275, respectively; more preferably, a molecule of the present disclosure has the amino acid sequence of any one of SEQ ID NOs: 156-167, 171-195, 202-204, 236-248, 253-256, 267, 270, and 272. Variants of the polypeptides of SEQ ID NOs: 156 to 167, 170 to 195, 202 to 205, 236 to 248, 253 to 256, and 266 to 275, preferably variants of any one of the polypeptides of SEQ ID NOs: 156 to 167, 171 to 195, 202 to 204, 236 to 248, 253 to 256, 267, 270, and 272, for example, variants of up to 50 amino acid modifications (e.g., amino acid substitutions (preferably and / or additions and / or deletions, for example up to 45, 40, 35, 30, 25 or 20 modifications, for example up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modification, or variants of SEQ ID NOs: 156 to 167, 170 to 195, 202 to 205, 236 to 248, 253 to 256 and 260, respectively. Also included are variants having at least 50% sequence identity to any one of the fusion polypeptides listed in Table 12A, such as SEQ ID NOs: 156-167, 171-195, 202-204, 236-248, 253-256, 267, 270, and 272, e.g., having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. It will be understood that variants must be such that the regulatory domain and targeting domain are still capable of performing their respective functions, such that the molecule can be targeted to a target substrate and the substrate can be consequently regulated.It will also be understood that the E2 ubiquitin or ubiquitin-like conjugate domain of the regulatory domain in a variant must still have at least 80% sequence identity with the human E2 enzyme or a functional portion thereof. It will be further understood that variants of the fusion polypeptides listed in Table 12A may have the targeting domain (e.g., aCS3 or K19 or a variant thereof) replaced with another targeting domain, such as aCS3 or K19 or a variant thereof, in some cases, and / or the regulatory domain replaced with another regulatory domain.

[0112] In some embodiments, the molecules of the present disclosure comprise a detectable marker, for example, to allow for identification or selection of cells containing the molecules of the present disclosure. The term "detectable marker" includes a marker that, when present in a molecule of the present disclosure, can be detected directly or indirectly, such that the presence of the molecule can also be detected. For example, it may be desirable to include a detectable marker in a fusion polypeptide of the present disclosure to determine whether the fusion polypeptide is being expressed. Thus, in one embodiment, a molecule of the present disclosure further comprises a detectable marker. Examples of detectable markers include affinity tags, such as a hemagglutinin A epitope tag (YPYDVPDYA; SEQ ID NO: 124), a Glu-Glu tag (CEEEEYMPME; SEQ ID NO: 125), and a FLAG tag (which binds to anti-FLAG antibodies). Other examples of markers that can be used are radioactive labels, fluorescent labels, enzyme labels, or other amino acid-based labels. While any suitable marker can be used, markers that do not contain lysine residues are preferred to minimize autoubiquitination, which can lead to proteolysis of the molecules of the present disclosure. Nucleic acid molecules encoding such peptide markers are available, for example, from Sigma-Aldrich Corporation (St. Louis, Mo., USA). It will be understood that the detectable marker can be present at the N-terminus of the fusion polypeptide or the C-terminus of the fusion polypeptide, or, if a peptide linker is present between the regulatory domain and the targeting domain, the detectable marker can be present within the linker of the fusion polypeptide. Examples of constructs with detectable markers at various positions can be found in Table 12A.

[0113] In further embodiments, the molecules of the present disclosure may include an additional localization moiety that can be used to direct the molecules of the present disclosure to a specific intracellular location. A localization moiety includes a moiety that targets the molecules of the present disclosure to a specific intracellular location, thereby increasing the concentration of the molecules of the present disclosure at that intracellular location compared to the concentration of the molecules of the present disclosure at that intracellular location in the absence of the localization moiety. The intracellular location may be a location where the target substrate is primarily present. For example, if the target substrate is primarily present in the nucleus, it may be desirable to include a localization moiety that directs the molecule to the nucleus. Similarly, it will be understood that the molecules of the present disclosure may be used to selectively regulate (e.g., degrade) a target substrate at a specific intracellular location. For example, a molecule may be used to regulate (e.g., degrade) a target substrate that is present in mitochondria but not the same substrate that is present in the nucleus.

[0114] Means for assessing subcellular localization are well known to those skilled in the art. For example, this can be tested by immunofluorescence staining and high-content imaging. Target proteins can be stained by staining with anti-tag antibodies to detect the presence of specific antibodies and molecules of the present disclosure. Fluorescently tagged secondary antibodies can be used to detect this by high-content confocal imaging. Furthermore, cell nuclei, mitochondria, or other organelles can be stained with specific dyes. Various localization motifs are known to those skilled in the art, including nuclear localization sequences (NLSs) that localize to the nucleus (Lange et al., J Biol Chem 2007, 282(8):5101-05), and CAAX motifs or palmitoylation sites that localize to the plasma membrane (Michaelson et al., Mol Biol Cell 2005, 16:1606-16; Guan and Fierke, Sci China Chem 2011, 54(12):1888-97; Aicart-Ramos et al., Biochim Biophys Acta - Biomembranes 2011, 1808(12):298-194). Any such localization motif may be included in the molecules of the present disclosure.

[0115] While the fusion polypeptides listed in Table 12A are shown in a particular orientation (e.g., "regulatory domain-linker-targeting domain" from N-terminus to C-terminus), for the avoidance of doubt, the reverse orientation is also within the scope of this disclosure. For example, the fusion polypeptide of SEQ ID NO: 193 (HA_UFC1_Linker2_aCS3) is in a "regulatory domain-linker-targeting domain" orientation, but it will be understood that the reverse "targeting domain-linker-regulatory domain" orientation is also within the scope of this disclosure.

[0116] Thus, it will be understood that the present disclosure provides fusion polypeptides comprising a regulatory domain, a targeting domain, optionally a peptide linker between the regulatory and targeting domains, and optionally a detectable marker and / or localization domain. For example, the present disclosure includes fusion polypeptides comprising a regulatory domain, a targeting domain, a peptide linker between the regulatory and targeting domains, and optionally a detectable marker and / or localization domain.

[0117] In a preferred embodiment, a first aspect of the present disclosure comprises a fusion polypeptide comprising an E2 enzyme having an amino acid sequence with at least 80% sequence identity to a human E2 enzyme (e.g., as listed in any of Tables 3-9 below) and a targeting domain (e.g., a monobody or nanobody) capable of targeting the E2 enzyme to a substrate.

[0118] In a preferred embodiment, the first aspect of the present disclosure comprises a fusion polypeptide comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a functional portion of a human E2 enzyme (e.g., as listed in any of Tables 3-9 below), and a targeting domain (e.g., a monobody or nanobody) capable of targeting the E2 enzyme to a substrate. Preferably, the functional portion is a UBC domain.

[0119] A second aspect of the present disclosure provides a compound comprising (i) a molecule according to the first aspect of the disclosure and (ii) a targeting moiety capable of targeting the molecule to a cell.

[0120] Preferences for molecules according to the first aspect of the present disclosure include those described above. For example, a compound may comprise a fusion polypeptide according to the first aspect of the present disclosure and a targeting moiety capable of targeting the molecule to a cell.

[0121] It will be understood that the cell is one that contains a target substrate that a molecule or polypeptide of the disclosure can modulate. Thus, the cell may contain a substrate that it is desired to degrade, and the molecule of the disclosure contains a regulatory domain that is a degradation domain.

[0122] By targeting moiety, we include the meaning of any moiety capable of targeting cells containing a substrate that is desired to be modulated (e.g., degraded). Cells containing a substrate that is desired to be modulated (e.g., degraded) include all cells containing the substrate, or a subset of cells containing the substrate, where it is desired to modulate (e.g., degrade) the substrate only in that subset of cells. Preferably, the targeting domain is capable of selectively targeting cells containing the substrate that is desired to be modulated (e.g., degraded). For example, the targeting moiety preferably targets cells to a greater extent than any other type of cell, and most preferably targets only cells containing the substrate that is desired to be modulated (e.g., degraded).

[0123] In one embodiment, the targeting moiety is a specific binding partner of an entity expressed by or associated with a cell containing a substrate desired to be modulated (e.g., degraded). Typically, the expressed entity is selectively expressed on the cell. For example, the abundance of the expressed entity is typically 10, 100, 500, 1000, 5000, or 10,000 higher on cells containing the substrate desired to be modulated (e.g., degraded) than on other cells, e.g., in the individual being treated.

[0124] The term "binding partner" includes the meaning of a molecule that binds to an entity expressed by a particular cell. Preferably, the binding partner selectively binds to that entity. For example, it is preferred if the binding partner has a Kd value (dissociation constant) that is at least 5-fold or 10-fold lower (i.e., higher affinity), preferably 100-fold or more than 500-fold lower, than at least one other entity expressed by another cell (e.g., a cell that does not contain the substrate that is desired to be modulated (e.g., degraded) or a cell that contains the substrate but that is not desired to be modulated (e.g., degraded) in that cell). More preferably, the binding partner for that entity has a Kd value that is 1000-fold or more than 5000-fold lower than at least one other entity expressed by another cell (e.g., a cell that does not contain the substrate that is desired to be modulated (e.g., degraded) or a cell that contains the substrate but that is not desired to be modulated (e.g., degraded) in that cell).

[0125] Typically, the binding partner binds to an entity that is present or accessible to the binding partner at a significantly higher concentration in or on cells containing the substrate desired to be modulated (e.g., degraded) than in any other cells of the host. Thus, the binding partner may bind to a surface molecule or antigen on a cell containing the substrate desired to be modulated (e.g., degraded) that is expressed in significantly higher amounts than on other cells. Similarly, the binding partner may bind to an entity secreted into the extracellular fluid by a cell containing the substrate desired to be modulated (e.g., degraded) to a greater extent than on other cells. For example, if the target substrate is present in a cancer cell, the binding partner may bind to a tumor-associated antigen expressed on the cell membrane or secreted into the extracellular fluid of the tumor.

[0126] In a preferred embodiment, the binding partner binds to an entity present or accessible to a cell containing the substrate desired to be modulated (e.g., degraded). Preferably, the entity, when bound by the binding partner, results in the internalization of the binding partner (and any associated molecules, e.g., compounds of the second aspect of the present disclosure) into the cell. If intracellular delivery relies on the endocytic pathway as the primary uptake mechanism, it will be appreciated that it may be desirable to include within the compound (e.g., a molecule of the first aspect of the present disclosure) a means of escape from endosomes or lysosomes, or any other vesicles that may be contained within endosomes or lysosomes, or to engage another means (i.e., external to the compound) to mediate such escape. Methods for enhancing endosomal escape are well known to those skilled in the art and include those reviewed in Hum Gen Ther 2011, 22(10):A14-A14 and in Loenn et al. (Sci Rep 2016, 6:32301). For example, the compound of the second aspect of the present disclosure (and / or the molecule of the first aspect of the present disclosure) may comprise an endosomal escape domain.

[0127] The targeting moiety can be either a polypeptide, peptide, small molecule or peptidomimetic. Typically, the targeting moiety is a polypeptide, such as any one of a monobody, nanobody, antibody, antibody fragment, scFv, intrabody, minibody, novel scaffold, peptide binder or ligand binding domain.

[0128] In a preferred embodiment, the targeting moiety is a binding partner such as an antibody. The antibody can bind to an antigen expressed by a cell containing the substance that is desired to be modulated (e.g., degraded), for example, an antigen expressed on the surface of the cell. Preferably, when bound by the antibody, the antigen causes the compound of the second aspect of the present disclosure to be internalized into the cell, for example, by receptor-mediated endocytosis.

[0129] It will be understood that when the molecules and targeting moieties of the present disclosure are polypeptides, the compounds of the second aspect of the present disclosure may also constitute a fusion polypeptide comprising a regulatory domain, a targeting domain, and a targeting moiety. Thus, the targeting moiety may be a polypeptide fused to a fusion polypeptide that itself comprises a targeting domain and a regulatory domain.

[0130] It will be appreciated that those skilled in the art can easily select an appropriate binding partner for any given cell type, for example, by identifying a surface antigen or molecule specific to that cell and finding a binding partner for that antigen or molecule. Considerable research has already been conducted on antibodies and fragments thereof against tumor-associated antigens, immune cell antigens, and infectious agents. Thus, in some embodiments, selecting an appropriate targeting moiety for a given cell type typically involves a literature search guided by, for example, the reviews of Muro ("Challenges in design and characterization of ligand-targeted drug delivery systems," J Control Release 2012, 164(2):125-37) and Carter et al. ("Identification and validation of cell surface antigens for antibody targeting in oncology," Endocr-relat Cancer 2004, 11:659-87). Alternatively, cells are obtained from a patient (e.g., by biopsy) and antibodies against those cells are prepared. Such "tailor-made" antibodies are already known. The antibodies have been demonstrated to confer binding to tumor cells not only in the patient from whom they were obtained, but also in many other patients, and therefore several such antibodies are commercially available.Other methods for identifying suitable binding partners for given unwanted cells include genetic approaches (e.g., microarrays), proteomic approaches (e.g., differential mass spectrometry), immunological approaches (e.g., immunizing animals with tumor cells and identifying antibody-secreting clones that specifically target the malignant cells), phage display selection using antibody libraries against the diseased cells themselves (phenotypic screening; see Rust et al., Mol Cancer 2013, 12:11, Sandercock et al., Mol Cancer 2015, 14:147 and Williams et al., Oncotarget 2016, 7(42)68278-91), and in silico approaches that use systems biology approaches to identify targets.

[0131] It will be understood that the targeting domain typically functions inside a cell to direct the regulatory domain to a target substrate (e.g., an intracellular polypeptide), while the targeting moiety typically functions outside a cell to target the regulatory domain and the targeting domain to that cell.

[0132] Antibody-drug conjugates for cancer therapy and the like have been reviewed by Carter & Senter (Cancer J 2008,14(3):154-69), and Chari et al. (Angewandte Chemie International Edition 2014, 53:3751), and it will be understood that compounds of this aspect of the disclosure can be considered to be such antibody-drug conjugates (see U.S. Pat. Nos. 5,773,001; 5,767,285; 5,739,116; 5,693,762; 5,585,089; U.S. Patent Application Publication No. 2006 / 0088522; U.S. Patent Application Publication No. 2011 / 0008840; U.S. Pat. No. 7,659,241; Hughes 2010 Nat Drug Discov 9:665, Lash 2010; In vivo The Business & Medicine Report 32-38; Mahato et al. 2011, Adv Drug Deliv Rev 63:659; Jeffrey et al. 2006, BMCL 16:358; see also Drugs RD 11(1):85-95). ADCs generally comprise a monoclonal antibody directed against a target present on tumor cells, a cytotoxic drug, and a linker connecting the antibody to the drug. Thus, the compound of the second aspect of the present disclosure may be an ADC comprising a targeting moiety that is an antibody, a regulatory domain, and a targeting domain. Preferences for the regulatory domain and the targeting domain include those described above in connection with the first aspect of the present disclosure.

[0133] The targeting moiety can be attached to the molecule of the first aspect of the present disclosure by known methods. For example, if the targeting moiety is a polypeptide such as an antibody and the molecule of the first aspect of the present disclosure is a fusion polypeptide, the targeting moiety, regulatory domain, and targeting domain can be expressed as a fusion polypeptide as known in the art and as described above. Alternatively, the targeting moiety can be attached to the molecule of the first aspect of the present disclosure by any other known means, either covalently or non-covalently.

[0134] In some embodiments, the targeting moiety is linked to the molecule of the present disclosure by a linker. By linker, we mean a chemical moiety that links the targeting moiety to the molecule of the first aspect of the present disclosure. The linkage can be covalent or non-covalent. Preferably, it is covalent. Thus, the targeting moiety and molecule of the first aspect of the present disclosure can be linked by any conventional method of cross-linking molecules, for example, as described above in connection with the first aspect of the present disclosure. It will be understood that numerous homobifunctional and heterobifunctional cross-linking chemistries are suitable for linking the targeting moiety to the T cell antigen, and any such chemistry can be used.

[0135] In some embodiments, the molecule of the first aspect of the present disclosure and the compound of the second aspect of the present disclosure are encoded by a suitable nucleic acid molecule and expressed in a suitable host cell. Thus, a third aspect of the present disclosure provides a polynucleotide encoding the molecule of the first aspect of the present disclosure or the compound of the second aspect of the present disclosure. Thus, when the molecule of the first aspect of the present disclosure or the compound of the second aspect of the present disclosure is a fusion polypeptide, it will be understood that the present disclosure includes a polynucleotide encoding such a fusion polypeptide. Preferences for the molecule of the first aspect of the present disclosure and the compound of the second aspect of the present disclosure include those described above for each aspect of the present disclosure.

[0136] A polynucleotide can be DNA or RNA. It can contain deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0137] Suitable nucleic acid molecules encoding the molecules of the first aspect of the present disclosure or the compounds of the second aspect of the present disclosure can be generated using standard cloning techniques, site-directed mutagenesis, and PCR, all of which are well known in the art. Molecular biology methods for cloning and manipulating genes and cDNA, mutating DNA, and expressing polypeptides from polynucleotides in host cells are well known in the art, as exemplified in "Molecular cloning, a laboratory manual," third edition, Sambrook, J. & Russell, DW (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference. Examples of suitable polynucleotides include those in Table 12C below, which have been assigned SEQ ID NOS: 223-235, 249-252, and 258-265. Preferably, the polynucleotide is any one of SEQ ID NOS: 223-235, 249-252, 259, 262, and 264.

[0138] A fourth aspect of the present disclosure provides a vector comprising the polynucleotide of the third aspect of the present disclosure. The vector can be of any type, for example, a recombinant vector such as an expression vector. The expression vector contains elements (e.g., a promoter, translation initiation and termination signals, and appropriate regions for transcriptional regulation) that allow the expression and / or secretion of a polypeptide in a host cell. Suitable expression systems include constitutive expression systems or inducible expression systems. In particular, the vector can be a viral vector, such as a lentivirus, adenovirus, or retrovirus. Most particularly, the vector can be a lentivirus or adeno-associated virus (AAV) vector. Other vectors include oncolytic viruses.

[0139] It is understood that in certain embodiments, the nucleic acid molecules and vectors may be used in the therapeutic aspects of the present disclosure via a gene therapy approach using formulations and methods described below and known in the art.

[0140] A fifth aspect of the present disclosure provides a host cell comprising a polynucleotide of the third aspect of the present disclosure or a polynucleotide of the fourth aspect of the present disclosure. Any of a variety of host cells can be used, including prokaryotic cells, such as E. coli, or eukaryotic cells, such as mammalian cells, human cells, yeast, insect cells, or plant cells. The host cell can be a cell line, such as a cancer cell line. Suitable examples of cells include Ad293, MDA-MB-231, U20S, HCT116, HeLa, and HEK293 cells. Many suitable vectors and host cells are well known in the art. Preferably, the host cell is a stable cell line. Alternatively, the host cell can be a cell obtained from a patient.

[0141] The present disclosure also includes methods for making the molecules of the first aspect of the disclosure or the compounds of the second aspect of the disclosure. For example, the present disclosure includes expressing a recombinant vector encoding the molecules of the present disclosure or the compounds of the first aspect or the second aspect of the disclosure in a suitable host cell and recovering the molecule or compound. Methods for expressing and purifying polypeptides are very well known in the art.

[0142] The present disclosure also provides a method of producing a cell, comprising introducing a polynucleotide molecule according to the third aspect of the present disclosure or a vector according to the fourth aspect of the present disclosure. Suitable methods of introducing the polynucleotide molecule and / or vector include those described above and are generally known in the art.

[0143] In addition to host cells used in methods for producing molecules or compounds of the present disclosure, the host cells themselves can be used directly in therapy, e.g., cell-mediated therapy. For example, it may be useful to selectively modulate or degrade disease-causing proteins, particularly their post-translational state (e.g., phosphorylation state), while still maintaining expression of other post-translational states. Thus, the present disclosure provides methods of treatment, including administering a host cell according to the present disclosure to a subject, for example, for use in medicine or to prevent or treat a disease or condition mediated by abnormal levels of the substrate or a form thereof in the subject. Thus, the present disclosure also provides host cells according to the fifth aspect of the present disclosure for use in medicine, e.g., for use in preventing or treating a disease or condition mediated by abnormal levels of the substrate or a form thereof in a subject. The present disclosure also provides the use of host cells in the manufacture of a medicament for use in medicine, e.g., a medicament for use in preventing or treating a disease or condition mediated by abnormal levels of the substrate or a form thereof in a subject. Foight et al., "Multi-input chemical control of protein dimerization for programming graded cellular responses," Nat Biotechnol 2019, 37(10):1209-16, describes the use of PROTACs in cell therapy. Further discussion of how various agents (e.g., molecules, compounds, polynucleotides, vectors, and compositions) of the present disclosure can be used therapeutically is provided below.

[0144] As explained below, a molecule of the disclosure or a compound of the disclosure may be clinically effective in the absence of any other therapeutic agent (e.g., an anti-cancer compound), although it may be advantageous to administer the molecule or compound (or a polynucleotide encoding the molecule or compound) in combination with an additional therapeutic agent.

[0145] Accordingly, a sixth aspect of the present disclosure provides a composition comprising a compound according to the first aspect of the present disclosure, a compound according to the second aspect of the present disclosure, a polynucleotide according to the third aspect of the present disclosure, a vector according to the fourth aspect of the present disclosure, or a cell according to the fifth aspect of the present disclosure, and a further therapeutic agent.

[0146] In one embodiment, the additional therapeutic agent is selected from the group consisting of anti-cancer agents, anti-viral agents, anti-diabetic agents, immunotherapeutic agents, anti-inflammatory agents, antibiotics, and any combination thereof. Examples of such agents are well known in the art and can be readily identified by one of ordinary skill in the art.

[0147] Preferably, the additional therapeutic agent is an anti-cancer agent, such as nitrogen mustards, such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; ethyleneimines and methylmelamines, such as hexamethylmelamine and thiotepa; alkylsulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin; and decarbazine (DTIC; dimethyl methacrylate). triazenes such as triazenoimidazole-carboxamides; antimetabolites, including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors, such as mercaptopurine (6-mercaptopurine; 6MP), thioguanine (6-thioguanine; TG) and pentostatin (2'- deoxycoformycin; natural products including vinca alkaloids, such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers, such as interferon ronalphenomenon; various drugs including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenediones such as mitoxantrone and anthracyclines; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogs / derivatives; cell cycle inhibitors; proteosome inhibitors such as bortezomib (Velcade®);The inhibitor may be selected from alkylating agents including signal transduction enzyme (e.g., tyrosine kinase) inhibitors such as imatinib (Glivec®), COX-2 inhibitors, and hormone agonists / antagonists such as flutamide and tamoxifen. In particular, tirapazamine may be utilized;

[0148] A seventh aspect of the present disclosure provides a molecule according to the first aspect of the disclosure, a compound according to the second aspect of the disclosure, a polynucleotide according to the third aspect of the disclosure, a vector according to the fourth aspect of the disclosure, a cell according to the fifth aspect of the disclosure or a composition according to the sixth aspect of the disclosure for use in medicine.

[0149] An eighth aspect of the present disclosure provides a pharmaceutical composition comprising a molecule according to the first aspect of the disclosure, a compound according to the second aspect of the disclosure, a polynucleotide according to the third aspect of the disclosure, a vector according to the fourth aspect of the disclosure, a cell according to the fifth aspect of the disclosure or a composition according to the sixth aspect of the disclosure, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0150] While the molecule described in the first aspect of the present disclosure, the compound described in the second aspect of the present disclosure, the polynucleotide described in the third aspect of the present disclosure, the vector described in the fourth aspect of the present disclosure, the cell described in the fifth aspect of the present disclosure, or the composition described in the sixth aspect of the present disclosure can be administered alone, it is preferable to provide it as a pharmaceutical formulation together with one or more acceptable carriers, diluents, or excipients. "Pharmaceutically acceptable" includes that the formulation is sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents, and excipients are well known in the pharmaceutical arts. The carrier(s) must be "acceptable" in the sense of being compatible with the inhibitor and not harmful to the recipient thereof. Typically, the carrier is sterile, pyrogen-free water or saline, although other acceptable carriers may be used.

[0151] Where appropriate, the formulations may be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (e.g., the molecule, compound, polynucleotide, vector, or composition of the present disclosure) with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0152] Formulations according to the present disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary, or paste.

[0153] In some embodiments, a unit dosage formulation is one containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of an active ingredient. It will be understood that in addition to the ingredients particularly mentioned above, the formulations of the present disclosure may include other agents conventional in the art having regard to the type of formulation in question; for example, those suitable for oral administration may include flavoring agents.

[0154] The amount of the agent of the present disclosure administered to an individual is an amount effective to combat the condition of the particular individual, which amount may be determined by a physician.

[0155] Preferably, in the context of any of the medical uses described herein, the subject to be treated is a human. Alternatively, the subject may be an animal, such as a domestic animal (e.g., a dog or cat), a laboratory animal (e.g., a laboratory rodent, such as a mouse, rat, or rabbit), or an animal of agricultural importance (i.e., livestock), such as a horse, cow, sheep, or goat.

[0156] It will be appreciated that molecules of the present disclosure can be delivered to cells (e.g., cells containing a substrate that is desired to be modulated (e.g., degraded)) in a variety of ways. For example, a molecule of the present disclosure can be a fusion polypeptide that is linked to a separate targeting moiety and thus can target cells of an individual, as described above with respect to the compounds of the second aspect of the present disclosure. In this manner, the fusion polypeptide can be brought into the vicinity of a cell and delivered to the cell, for example, by internalization after the targeting moiety binds to an entity on the cell (e.g., on the cell surface). Alternatively, a molecule of the present disclosure can be a fusion polypeptide and delivered to a cell by introducing a polynucleotide or vector encoding the fusion polypeptide into the cell.

[0157] Accordingly, a ninth aspect of the present disclosure provides a method of delivering a molecule according to the first aspect of the present disclosure to a cell of an individual (e.g., a cell containing a substrate that it is desired to modulate (e.g., degrade)), the method comprising administering to the individual a compound of the second aspect of the present disclosure, or administering to the individual a polynucleotide of the third aspect of the present disclosure or a vector of the fourth aspect of the present disclosure, wherein the polynucleotide or vector encodes the molecule in the cell.

[0158] The molecules, compounds, polynucleotides or vectors may be administered orally or by any parenteral route, for example, in the form of a pharmaceutical preparation containing the active ingredient, optionally in the form of a non-toxic organic or inorganic acid or base addition salt in a pharmaceutically acceptable dosage form. The active ingredient may be administered in various doses.

[0159] The present disclosure also provides a compound of the second aspect of the present disclosure, a polynucleotide of the third aspect of the present disclosure, or a vector of the fourth aspect of the present disclosure for use in delivering a molecule of the first aspect of the present disclosure to a cell of an individual (e.g., a cell containing a substrate that it is desired to modulate (e.g., degrade)).

[0160] Similarly, the present disclosure also provides the use of a compound according to the second aspect of the disclosure, a polynucleotide according to the third aspect of the disclosure, or a vector according to the fourth aspect of the disclosure in the manufacture of a medicament for delivering a molecule according to the first aspect of the disclosure to a cell of an individual (e.g., a cell containing a substrate that it is desired to modulate (e.g., degrade)).

[0161] A tenth aspect of the present disclosure provides a kit-of-parts comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence having at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; and (b) a targeting domain capable of targeting the regulatory domain to a substrate; optionally, the kit does not comprise an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof.

[0162] Preferences for the regulatory domain, E2 ubiquitin or ubiquitin-like conjugating domain, targeting domain, substrate and E3 ubiquitin or ubiquitin-like ligase or functional portion thereof include those described above with respect to the first aspect of the disclosure.

[0163] In one embodiment, the kit further comprises a linking means suitable for linking the regulatory domain to the targeting domain. Any suitable linking means can be used, including linkers as described elsewhere herein. Thus, the kit may further comprise a linker capable of joining the regulatory domain to the targeting domain. The linkage can be covalent or non-covalent.

[0164] In a further embodiment, the kit further comprises a targeting moiety capable of targeting cells containing the substrate to be modulated (e.g., degraded). It will be appreciated, therefore, that the kit may be useful in a "plug-and-play" context, in which appropriate regulatory domains, targeting domains, and targeting moieties are selected and then combined to form a treatment tailored to a given individual. It will be appreciated that such kits are suitable for use in the therapeutic aspects of the present disclosure described herein and below. For example, if a cancer is shown to be dependent on the expression or activity of a particular oncogene, that oncogene can be targeted for degradation or other modulation. This can be achieved using a promiscuous E2 enzyme or a functional portion or variant thereof; however, if the oncogene is known to be a substrate protein for a particular E2 enzyme, that E2 enzyme may be selected. Preferences for the targeting moiety include those described above with respect to the second aspect of the present disclosure. Preferably, the targeting moiety is an antibody.

[0165] An eleventh aspect of the present disclosure provides a kit-of-parts comprising: (a) a molecule of the first aspect of the present disclosure; and (b) a targeting moiety capable of targeting a cell containing a substrate to be modulated (e.g., degraded). Preferences for the molecule and substrate of the first aspect of the present disclosure include those described above with respect to the first aspect of the present disclosure, and preferences for the targeting moiety include those described above with respect to the second aspect of the present disclosure. Preferably, the targeting moiety is an antibody. Again, it will be understood that such kits are suitable for use in the therapeutic aspects of the present disclosure described herein and below and may be useful in a "plug-and-play" context.

[0166] In one embodiment, the kit further comprises a linking means suitable for linking the molecule of the first aspect of the present disclosure to a targeting moiety. Any suitable linking means can be used, including linkers as described elsewhere herein. Thus, the kit may further comprise a linker capable of linking the molecule of the first aspect of the present disclosure to a targeting moiety. The linking may be covalent or non-covalent.

[0167] A twelfth aspect of the present disclosure provides a kit-of-parts comprising: (a) a polynucleotide encoding a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence having at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; and (b) a polynucleotide encoding a targeting domain capable of targeting the regulatory domain to a substrate; optionally, the kit does not comprise a polynucleotide encoding an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof.

[0168] Preferences for the regulatory domain, E2 ubiquitin or ubiquitin-like conjugating domain, targeting domain, substrate and E3 ubiquitin or ubiquitin-like ligase or functional portion thereof include those described above with respect to the first aspect of the disclosure.

[0169] In embodiments, the kit includes one or more promoter sequences capable of directing expression of one or both of the polynucleotides in cells containing the regulated substrate. The promoters may be constitutively active or inducible, thereby allowing for temporal regulation of expression of the polynucleotides in the cells. It may be useful to use tissue-specific promoters to target expression to particular cell types or tissues. Such promoters are well known in the art and can be readily sourced or designed, for example, based on review of the scientific literature.

[0170] A further kit-of-parts provided by the present disclosure includes (a) a polynucleotide encoding a molecule according to the first aspect of the present disclosure and (b) a targeting moiety capable of targeting cells containing a substrate to be modulated. Preferences for the molecule according to the first aspect of the present disclosure and the targeting moiety include those described above. It will be understood that such kits can also be used in a "plug-and-play" system as described above, where a polynucleotide encoding a molecule according to the first aspect of the present disclosure can be used to express such a molecule that can be conjugated to an appropriate targeting moiety, e.g., depending on the ultimate therapeutic application.

[0171] As discussed below, the agents of the present disclosure are useful for preventing or treating diseases or conditions mediated by abnormal levels of a substrate in a subject. Therefore, it will be appreciated that it may be useful to identify or confirm which substrates are at abnormal levels in cells, such as cells in a biopsy sample taken from the subject, before treating the subject. It will be appreciated that any of the kits of parts described above may be useful if they further include one or more reagents for assessing the expression profile of cells containing the substrate to be modulated. Assessment of the expression profile of cells (e.g., in a biopsy sample) can be performed using routine assays for measuring nucleic acid (e.g., DNA or RNA transcripts) or protein levels. For example, transcriptomic or proteomic techniques can be used. Any suitable reagents can be used, including a binding partner for the nucleic acid encoding the substrate, a binding partner for the substrate itself, and PCR primers. Preferably, the reagent is an antibody that binds to the substrate.

[0172] As discussed further below, the disclosed agents have utility in assessing substrate function by assessing the effect of the disclosed molecules on one or more properties of a cell, tissue, or organ. Thus, in some embodiments, any of the kits-of-parts described above can further include a means for assessing a cellular property. In this manner, the kit can be used in a screening context, for example, to identify a substrate that has a particular effect on a given cellular property.

[0173] The cellular characteristics we refer to include any of survival, growth, proliferation, differentiation, migration, morphology, signal transduction, metabolic activity, gene expression, protein translation, and cell-cell interactions. Evaluating one or more cellular characteristics can be performed using any suitable method known in the art. For example, any of cell survival, growth, proliferation, differentiation, migration, and morphology can be evaluated by microscopy or image analysis. Characteristics can also be detected using appropriate markers. For example, the expression of detectably labeled proteins, reporters, and / or one-step labeling of cellular components and markers can enable direct visualization of cellular structure, multicellular organization, and other readouts, for example, by fluorescence microscopy (e.g., E-cadherin staining to identify cell-cell contacts). Gene expression can be evaluated by functional genomics (e.g., microarray) techniques, proteomics techniques, or protein translation by immunohistochemistry. Immunofluorescence, Hoeschst staining, or Annexin V assays can be used. Therefore, it is understood that those skilled in the art can select an appropriate technique and, thereby, an appropriate means for evaluating a given characteristic. Examples of means include antibodies, primers, enzyme reagents, immunoassay reagents, or any detectable marker (eg, protein or nucleic acid) of the entity of interest.

[0174] A thirteenth aspect of the present disclosure provides a method of preventing or treating a disease or condition mediated by abnormal levels of a substrate or a form thereof in a subject, the method comprising administering to the subject a molecule of the first aspect of the present disclosure, a compound of the second aspect of the present disclosure, a polynucleotide of the third aspect of the present disclosure, a vector of the fourth aspect of the present disclosure, a cell according to the fifth aspect of the present disclosure, a composition of the sixth aspect of the present disclosure, and a pharmaceutical composition of the eighth aspect of the present disclosure.

[0175] Similarly, the present disclosure provides a molecule of the first aspect of the present disclosure, a compound of the second aspect of the present disclosure, a polynucleotide of the third aspect of the present disclosure, a vector of the fourth aspect of the present disclosure, a cell according to the fifth aspect of the present disclosure, a composition of the sixth aspect of the present disclosure, and a pharmaceutical composition of the eighth aspect of the present disclosure for use in preventing or treating a disease or condition mediated by abnormal levels of the substrate or a form thereof in a subject.

[0176] Similarly, the present disclosure provides use of a molecule of the first aspect of the present disclosure, a compound of the second aspect of the present disclosure, a polynucleotide of the third aspect of the present disclosure, a vector of the fourth aspect of the present disclosure, a composition of the sixth aspect of the present disclosure, and a pharmaceutical composition of the eighth aspect of the present disclosure in the manufacture of a medicament for preventing or treating a disease or condition mediated by abnormal levels of a substrate or a form thereof in a subject.

[0177] By preventing or treating a condition, we include meaning to alleviate or relieve symptoms in a patient (i.e., palliative uses), to prevent symptoms from worsening or progressing, to treat a disorder (e.g., by inhibiting or removing a causative agent), or to prevent a condition or disorder in a subject who is not free of the condition or disorder.

[0178] "Condition mediated by abnormal levels of a substrate or a form thereof" includes any biological or medical condition or disorder in which at least part of the pathology is mediated by abnormal levels of a substrate or a form thereof. The condition may be caused by abnormal levels of a substrate or a form thereof, or the abnormal levels of that form of the substrate may be an effect of the condition. Abnormal levels include the meaning that the substrate or a form thereof is present at a higher or lower level than the substrate or a form thereof in a normal, non-pathological state. It will be understood that while the amount of the substrate itself may remain the same between a pathological and a non-pathological state, the proportion of the amount of the substrate present in a particular form (e.g., a particular post-translationally modified form) may be higher or lower in a pathological state. For the avoidance of doubt, by abnormal levels of a substrate, we include the meaning of abnormal levels of a form of that substrate, for example, a post-translationally modified form (e.g., a phosphorylated form). Examples of specific conditions include cancer, diabetes, autoimmune diseases, Alzheimer's disease, Parkinson's disease, pain, viral diseases, bacterial diseases, prion diseases, fungal diseases, parasitic diseases, arthritis, immune deficiencies, and inflammatory diseases.

[0179] Agents of the present disclosure (e.g., molecules of the first aspect of the present disclosure, compounds of the second aspect of the present disclosure, polynucleotides of the third aspect of the present disclosure, vectors of the fourth aspect of the present disclosure, cells of the fifth aspect of the present disclosure, compositions of the sixth aspect of the present disclosure, and pharmaceutical compositions of the eighth aspect of the present disclosure) may be formulated in any suitable manner and / or administered to an individual by any suitable route of administration and / or at an appropriate dose, for example, as described above and as determined by a physician.

[0180] A fourteenth aspect of the present disclosure provides a method of modulating a substrate, comprising contacting the substrate with a molecule of the first aspect of the present disclosure under conditions effective for the molecule to modulate the substrate. Preferences for the molecule and substrate of the first aspect of the present disclosure include those described above.

[0181] By modulating, we include meaning any possible type of modulation that can be mediated by ubiquitin or ubiquitin-like proteins, for example, modulating one or more activities of a target substrate, and / or modulating the cellular location of a target substrate, and / or modulating the stability of a target substrate. Preferably, modulating includes degrading the substrate. Thus, in embodiments, modulating includes degrading the substrate, or preventing the substrate from being degraded, or altering the subcellular location of the substrate, or modulating (e.g., increasing or decreasing) one or more activities of the substrate, or modulating the degree of post-translational modification of the substrate. This method can be performed in vivo or in vitro.

[0182] "Under conditions effective for the molecule to modulate the substrate" includes the meaning that the substrate is contacted with a molecule of the present disclosure under conditions that allow the formation of a complex between the substrate and the molecule such that ubiquitin or ubiquitin-like protein can be conjugated to the substrate, thereby modulating the substrate. Minimal conditions would be the presence of an E1 protein, ubiquitin or ubiquitin-like protein, and the cellular machinery for the specific regulation mediated by ubiquitin or ubiquitin-like protein. For example, if the specific regulation is ubiquitin-mediated degradation, then effective conditions for the molecule to degrade the substrate would include the cellular machinery necessary for such degradation, such as the proteasome. Typically, the method is performed intracellularly, and therefore the cellular conditions are effective for the molecule to modulate the substrate. However, in vitro ubiquitination assays are known, and thus the method can be performed in vitro, for example, to further understand the mechanism, kinetics, and location of ubiquitin or ubiquitin-like protein attachment.

[0183] It is understood that the agents of the present disclosure are useful for identifying and / or validating substrates as potential drug targets. Because the agents of the present disclosure provide targeted modulation (e.g., degradation) of cellular substrates, including intracellular substrates, the effects of such modulation can be beneficial in therapeutic settings.

[0184] Accordingly, a fifteenth aspect of the present disclosure is a method for identifying a substrate as a potential drug target, comprising: (a) providing a cell, tissue, or organ comprising a substrate; (b) contacting the cell, tissue or organ with a molecule according to the first aspect of the present disclosure, or a compound according to the second aspect of the present disclosure, or a polynucleotide according to the third aspect of the present disclosure, or a vector according to the fourth aspect of the present disclosure; (c) assessing the effect of the molecule, compound, polynucleotide or vector on one or more properties of a cell, tissue or organ, wherein identification of an effect that correlates with a particular disease state indicates that the substrate is a potential drug target for the particular disease.

[0185] Suitable cells, or tissues / organs from which they can be derived, include bone marrow, skin, cartilage, tendon, bone, muscle (including cardiac muscle), blood vessel, cornea, nerve, brain, gastrointestinal, kidney, liver, pancreas (including pancreatic islet cells), lung, pituitary, thyroid, adrenal, lymph, saliva, ovary, testis, cervix, bladder, endometrium, prostate, vulva, and esophagus. Also included are various cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. The cells can be stem cells, progenitor cells, or somatic cells. Preferably, the cells are mammalian cells, such as human cells, or cells derived from animals, such as mice, rats, or rabbits. It is understood that the cells can be derived from normal or healthy biological tissues, or from diseased or affected biological tissues, such as tissues or fluids derived from tumors.

[0186] It will be appreciated that the methods may be performed in vivo, ex vivo, or in vitro. For example, the methods may be performed on tissues or organs ex vivo, on cell culture in vitro, or on cells, tissues, or organs when in their natural environment in vivo.

[0187] It will be appreciated that the molecule of the first aspect of the present disclosure may be delivered to a cell, organ or tissue by direct contact with a molecule of the first aspect of the present disclosure or a compound of the second aspect of the present disclosure (e.g., where the compound comprises a targeting moiety that binds to an entity on the surface of the cell and results in internalization of the compound), or by expressing a polynucleotide of the third aspect of the present disclosure or a vector of the fourth aspect of the present disclosure.

[0188] By assessing the effect of a molecule, compound, polynucleotide, or vector on one or more properties of a cell, tissue, or organ, we mean assessing the effect on any one or more properties of a cell, tissue, or organ known to correlate with a particular disease state. In this way, by knowing that modulation (e.g., degradation) of a substrate affects one or more properties, it is possible to identify that substrate as a potential drug target for a particular disease.

[0189] Any characteristic of a cell, tissue, or organ can be evaluated, and for a given disease or condition, one skilled in the art would be able to readily identify the appropriate characteristic to evaluate. Thus, the one or more characteristics can be any of the cellular characteristics described above in connection with the twelfth aspect of the present disclosure, such as characteristics selected from the group consisting of survival, growth, proliferation, differentiation, migration, morphology, signal transduction, metabolic activity, gene expression, protein translation, and cell-cell interactions. Characteristics of tissues and organs include morphology and multicellular organization. In the context of cancer, the characteristics evaluated can include any one or more of cell growth, proliferation, differentiation, and migration.

[0190] Assessing one or more properties of the cells, tissues, or organs can be performed using any suitable method known in the art, for example, as described above in connection with the twelfth aspect of the present disclosure. In some embodiments, the method can be performed using one of the kits-of-parts of the present disclosure described above.

[0191] In a manner similar to the fifteenth aspect of the present disclosure, it will be appreciated that the agents of the present disclosure may be useful for assessing the function of substrates and the effect of their upregulation, for example by degrading the substrates and assessing the effect, or by otherwise modulating the substrates.

[0192] Accordingly, a sixteenth aspect of the present disclosure is a method for assessing the function of a substrate, comprising: (a) providing a cell, tissue, or organ comprising a substrate; (b) contacting the cell, tissue or organ with a molecule according to the first aspect of the present disclosure, or a compound according to the second aspect of the present disclosure, or a polynucleotide according to the third aspect of the present disclosure, or a vector according to the fourth aspect of the present disclosure; (c) assessing the effect of the molecule, compound, polynucleotide or vector on one or more properties of the cell, tissue or organ.

[0193] The preferences of the cells, tissues and organs and the one or more properties of the cells, tissues or organs include those described above in relation to the fifteenth aspect of the present disclosure. It will be understood that the method can be performed in vivo, ex vivo, or in vitro. For example, the method can be performed on tissues or organs ex vivo, on cell cultures in vitro, or on cells, tissues, or organs in their natural environment in vivo. It will also be understood that the method allows for assessment of cellular gene or protein function, for example, when the substrate is a protein encoded by the gene. In some embodiments, the method can be performed using one of the kits of parts of the present disclosure described above.

[0194] A seventeenth aspect of the present disclosure is a method for identifying an agent that may be useful in preventing or treating a disease or condition mediated by abnormal levels of a substrate or form thereof, the method comprising: providing a substrate; providing a test agent comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugate domain having an amino acid sequence with at least 80% sequence identity to a human E2 ubiquitin or ubiquitin-like domain; and (b) a targeting domain capable of targeting the regulatory domain to a substrate, wherein optionally the test agent does not comprise an E3 ubiquitin or ubiquitin-like ligase or portion thereof; contacting the substrate and the test agent under conditions effective for the test agent to facilitate conditioning of the substrate; and determining whether the test agent modulates the substrate.

[0195] Preferences for substrates and diseases or conditions mediated by abnormal levels of the substrate or its form include those described above. For example, the substrate (e.g., protein) can be an intracellular protein that, itself or a form thereof (e.g., a post-translationally modified form such as a phosphorylated form) is involved in a particular disease or condition.

[0196] It will be appreciated that the test agent may be a molecule according to the first aspect of the present disclosure. Thus, the method may be used to assess the effectiveness of a candidate molecule of the first aspect of the present disclosure to modulate a substrate (e.g., degrade the substrate), thereby identifying the molecule as one that may be useful in combating a disease or condition mediated by abnormal levels of the substrate or a form thereof.

[0197] It will be appreciated that the methods may be performed in vivo, ex vivo, or in vitro. For example, the methods may be performed on tissues or organs ex vivo, on cell culture in vitro, or on cells, tissues, or organs when in their natural environment in vivo.

[0198] "Conditions effective for the test agent to promote modulation of the substrate" includes the meaning that the substrate is contacted with a molecule of the present disclosure under conditions that allow the formation of a complex between the substrate and the molecule such that ubiquitin or a ubiquitin-like protein can be conjugated to the substrate, thereby modulating the substrate. Minimum conditions include those defined above with respect to the fourteenth aspect of the present disclosure. It is preferred that the method is performed intracellularly, and thus the cellular conditions are effective for the test agent to promote modulation of the substrate. However, in vitro ubiquitination assays are known, and thus the method can be performed in vitro.

[0199] In a preferred embodiment, the test agent is one that degrades a substrate. It is understood that in some instances, high-throughput screening of test agents will be preferred, and that the method may be used as a "library screening" method, a term well known to those skilled in the art. Thus, the test agent may be a library of test agents. Methods for preparing and screening such libraries are known in the art.

[0200] The present disclosure includes screening methods for identifying drugs or lead compounds for use in treating a disease or condition. It is understood that screening assays that are amenable to high-throughput operation are particularly preferred. It is understood that identification of a test agent that modulates (e.g., degrades) a substrate may be the first step in a drug screening pathway, and that the agent may be further selected and / or further modified, for example, based on its effectiveness in an assay for the disease or condition of interest. Thus, the method may further include a step of testing the test agent in an assay for the disease or condition of interest. Assays for various diseases and conditions are known in the art.

[0201] The method may include the further step of synthesizing and / or purifying the identified agent or modified agent. The present disclosure may further include the step of synthesizing, purifying, and / or formulating the identified test agent. The agent may also be subjected to other tests, such as toxicology or metabolic tests, as known to those skilled in the art. The present disclosure includes the use of the molecule of the first aspect of the present disclosure, or the compound of the second aspect of the present disclosure, or the polynucleotide of the third aspect of the present disclosure, or the vector of the fourth aspect of the present disclosure in drug target validation or drug discovery.

[0202] In the foregoing description, particular embodiments may be described in isolation for clarity. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless expressly specified otherwise that the features of a particular embodiment are incompatible with the features of another embodiment.

[0203] For any method disclosed herein that includes discrete steps, the steps may be performed in any practicable order, and, where appropriate, any combination of two or more steps may be performed simultaneously.

[0204] All documents mentioned herein are incorporated herein by reference in their entirety. The listing or discussion of a document herein that appears to be prior-published should not be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The present disclosure relates, for example, to the following: [1] A molecule, (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; (b) a targeting domain capable of targeting the regulatory domain to a substrate; and Contains, molecule. [2] The molecule according to [1] above, which does not contain an E3 ubiquitin or ubiquitin-like ligase or a functional part thereof. [3] The molecule according to [1] or [2], wherein the regulatory domain comprises an E2 ubiquitin conjugate domain capable of binding to ubiquitin and transferring the ubiquitin to the substrate, or the regulatory domain comprises an E2 ubiquitin-like conjugate domain capable of binding to a ubiquitin-like protein and transferring the ubiquitin-like protein to the substrate. [4] The molecule described in [3] above, wherein the ubiquitin-like protein is SUMO, NEDD8, ATG8, ATG12, ISG15, UFM1, FAT10, URM1, or FUBI. [5] The molecule according to any one of [1] to [4] above, wherein the E2 ubiquitin or ubiquitin-like conjugate domain comprises a ubiquitin core catalytic (UBC) domain. [6] The molecule according to [5], wherein the UBC domain comprises 110 to 290 amino acids, such as 117 to 284 amino acids or 140 to 192 amino acids. [7] The molecule according to any one of [1] to [6] above, wherein the UBC domain contains a catalytic cysteine ​​residue. [8] The molecule according to any one of [5] to [7], wherein the UBC domain comprises a PxxxP (SEQ ID NO: 206) peptide motif and a tryptophan residue located 26 to 43 amino acids from the C-terminus of the PxxxP motif, and optionally the PxxxP peptide motif is PxxPP (SEQ ID NO: 207). [9] The molecule according to any one of [5] to [8], wherein (i) the UBC comprises an HxN peptide motif, and optionally the HxN motif is an HPN tripeptide, or (ii) the UBC comprises a TxNGRF (SEQ ID NO: 210) peptide motif, and optionally the TxNGRF peptide motif is TPNGRF (SEQ ID NO: 208) or TANGRF (SEQ ID NO: 209).

[10] The molecule according to any one of [1] to [9] above, wherein the E2 ubiquitin or ubiquitin-like conjugate domain is derived from an E2 enzyme or is synthetic.

[11] The molecule according to any one of [1] to

[10] above, wherein the regulatory domain comprises an E2 enzyme containing an E2 ubiquitin or ubiquitin-like conjugate domain.

[12] The molecule according to

[10] or

[11] , wherein the E2 enzyme is a family 1 E2 enzyme, family 2 E2 enzyme, family 3 E2 enzyme, family 4 E2 enzyme, family 5 E2 enzyme, family 6 E2 enzyme, family 7 E2 enzyme, family 8 E2 enzyme, family 9 E2 enzyme, family 10 E2 enzyme, family 11 E2 enzyme, family 12 E2 enzyme, family 13 E2 enzyme, family 14 E2 enzyme, family 15 E2 enzyme, family 16 E2 enzyme, or family 17 E2 enzyme.

[13] The molecule according to any one of

[10] to

[12] above, wherein the E2 enzyme is a class I E2 enzyme, a class II E2 enzyme, a class III E2 enzyme, or a class IV E2 enzyme.

[14] The molecule according to any one of

[10] to

[13] above, wherein the E2 enzyme has an amino acid sequence that has at least 85%, 90%, 95%, 99%, or 100% sequence identity to human E2 enzyme.

[15] The E2 enzyme is UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2 D2(UbcH5B), UBE2D3(UbcH5C), UBE2D4(HBUCE1), UBE2E1(UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F(NCE2), UBE2G1(UBE2G), UBE2G2(UBC7), UBE2H(UBCH), UBE2I(Ubc9), UBE2J1(NCUBE1), UBE2J2(NCUBE2), UBE2K(HIP2), UBE2L3(UbcH7), UBE2L6(Ub cH8), UBE2M (Ubc12), UBE2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, or UFC1.

[16] The molecule according to any one of

[10] to

[15] above, wherein the E2 enzyme is UBE2D1 (UbcH5A), UBE2E2, UBE2L3 (UbcH7), UBE2O (E2-230K), UBE2Q2, or UBE2R2.

[17] The E2 ubiquitin or ubiquitin-like conjugate domain is selected from the group consisting of UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E 3(UbcH9), UBE2F(NCE2), UBE2G1(UBE2G), UBE2G2(UBC7), UBE2H(UBCH), UBE2I(Ubc9), UBE2J1 (NCUBE1), UBE2J2(NCUBE2), UBE2K(HIP2), UBE2L3(UbcH7), UBE2L6(UbcH8), UBE2M(Ubc12), UB The molecule according to any one of [1] to

[16] above, comprising a UBC domain having an amino acid sequence that is at least 80% identical to the UBC domain of any one of E2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1 (SEQ ID NOs: 42 to 82, respectively).

[18] The E2 ubiquitin or ubiquitin-like conjugate domain is selected from the group consisting of UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE 2E3(UbcH9), UBE2F(NCE2), UBE2G1(UBE2G), UBE2G2(UBC7), UBE2H(UBCH), UBE2I(Ubc9), UBE 2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (UbcH7), UBE2L6 (UbcH8), UBE2M (Ubc1 2), UBE2N (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1, and the amino acid sequences of the UBC domains are identified by SEQ ID NOs: 42 to 82, respectively.

[19] The regulatory domains are UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NC E2), UBE2G1 (UBE2G), UBE2G2 (UBC7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K(HIP2), UBE2L3(UbcH7), UBE2L6(UbcH8), UBE2M(Ubc12), UBE2N(Ubc13), UB The molecule according to any one of [1] to

[18] above, comprising an E2 enzyme having an amino acid sequence that has at least 80% sequence identity to any one of the E2 enzymes selected from the group consisting of E2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1 (SEQ ID NOs: 1 to 41, respectively).

[20] The regulatory domain is selected from the group consisting of UBE2A (hHR6A), UBE2B (hHR6B), UBE2C (UbcH10), UBE2D1 (UbcH5A), UBE2D2 (UbcH5B), UBE2D3 (UbcH5C), UBE2D4 (HBUCE1), UBE2E1 (UbcH6), UBE2E2, UBE2E3 (UbcH9), UBE2F (NCE2), UBE2G1 (UBE2G), UBE2G2 (UBC7), UBE2H (UBCH), UBE2I (Ubc9), UBE2J1 (NCUBE1), UBE2J2 (NCUBE2), UBE2K (HIP2), UBE2L3 (UbcH7), UBE2L6 (UbcH8), UBE2M (Ubc12), and UBE2N. (Ubc13), UBE2NL, UBE2O (E2-230K), UBE2Q1 (NICE-5), UBE2Q2, UBE2QL, UBE2R1 (CDC34), UBE2R2 (CDC34B), UBE2S (E2-EPF), UBE2T (HSPC150), UBE2U, UBE2V1 (UEV-1A), UBE2V2 (MMS2), UBE2W, UBE2Z (Use1), UVELD (UEV3), BIRC6 (apollon), FTS (AKTIP), TSG101, and UFC1, and the amino acid sequences of the E2 enzymes are set forth in SEQ ID NOs: 1 to 41, respectively. [twenty one] The molecule according to any one of [1] to

[20] above, wherein the targeting domain binds to the substrate. [twenty two] The molecule according to any one of [1] to

[21] , wherein the targeting domain is any one of a monobody, a nanobody, an antibody, an antibody fragment, an scFv, an intrabody, a minibody, a scaffold protein, such as a designed ankyrin repeat protein (DARPin), a peptide binder, and a ligand-binding domain. [twenty three] The molecule according to any one of [1] to

[22] above, wherein the targeting domain and / or the regulatory domain does not contain a lysine residue. [twenty four] The molecule according to any one of [1] to

[23] above, wherein the targeting domain has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 126 to 135, 138 to 139, and 257, and / or the regulatory domain has an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1 to 82. [twenty five] The molecule according to any one of [1] to

[24] above, wherein the targeting domain has the amino acid sequence of any one of SEQ ID NOs: 126 to 135, 138 to 139, and 257, or a variant thereof having up to 20 amino acid modifications, and / or the regulatory domain has the amino acid sequence of any one of SEQ ID NOs: 1 to 82, or a variant thereof having up to 30 amino acid modifications.

[26] the targeting domain is a variant of the amino acid sequence of any one of SEQ ID NOs: 126-135, 138-139, 257, in which one or more of the lysine residues have been substituted with another amino acid and / or deleted; and / or The regulatory domain is a variant of the amino acid sequence of any one of SEQ ID NOs: 42 to 82, in which one or more lysine residues are substituted with another amino acid and / or deleted. The molecule according to any one of

[23] to

[25] above.

[27] The molecule according to any one of [1] to

[26] above, wherein the substrate is an intracellular polypeptide.

[28] The molecule according to any one of [1] to

[27] above, wherein the substrate is localized in one or more of the plasma membrane, cytoplasm, nucleus, endosome, endoplasmic reticulum, mitochondria, and Golgi apparatus.

[29] The molecule according to any one of [1] to

[28] above, wherein the substrate is localized in the nucleus.

[30] The molecule according to any one of [1] to

[28] above, wherein the substrate is an oncogenic protein, a signal transduction protein, a GPCR, a post-translational modification protein, an adhesion protein, a receptor, a cell cycle protein, a checkpoint protein, a viral protein, a prion protein, a bacterial protein, a parasitic protein, a fungal protein, a DNA-binding protein, a structural protein, an enzyme, an immunogen, an antigen, and / or a pathogenic protein.

[31] The molecule according to any one of [1] to

[30] above, wherein the substrate is selected from the group consisting of Ras, KRas, SHP2, human rhinovirus (HRV) protease 3C, muscarinic acetylcholine receptor 2 (M2R), beta-2 adrenergic receptor (β2-AR), cross-binding endonuclease MUS81 (MUS81), and human antigen R (HuR).

[32] The molecule according to any one of [1] to

[31] above, wherein the regulatory domain and the targeting domain are linked by a linker.

[33] 32. The molecule according to claim 32, wherein the linker is a polypeptide linker, for example a polypeptide containing one or more glycine and / or serine amino acid residues.

[34] The molecule according to

[31] or

[32] , wherein the linker is 1 to 45 amino acids in length, for example, 6 to 20 amino acids in length or 5 to 19 amino acids in length.

[35] The linker is selected from the group consisting of the peptides GGGGS (SEQ ID NO: 146), GGGGSGGGSGGGGS (SEQ ID NO: 145), LEGGGGSSR (SEQ ID NO: 141), LEGGGGSGGGGSGGGGSSR (SEQ ID NO: 142), AAAGGGGSGGGGSGGGGSGT (SEQ ID NO: 143), GGGGG (SEQ ID NO: 144), LEGGSR (SEQ ID NO: 211), LEGGGSGGSSR (SEQ ID NO: 212), LEGGGGSGGGSSR (SEQ ID NO: 213), LEGGGSGGGSGGGSSR (SEQ ID NO: 214), LEGGGGSGPSGGGGPSGSR (SEQ ID NO: 215), LESNGGGGSPA The molecule according to any one of

[32] to

[34] , comprising PAPGGGGSGSSR (SEQ ID NO: 216), LEGGGGSYPYDVPDYASGGGGSSR (SEQ ID NO: 217), TGGSAGGSGGSAGGSGGSAGGSGGSA (SEQ ID NO: 218), AGSGGSTGSGGSPTPSTSGGSTGSGGAS (SEQ ID NO: 219), AGSGGSGGSGGSGNSSTSGGSGGSGGAS (SEQ ID NO: 220), GGSPVPSTPGGGSGGGSGGSPVPSTPGS (SEQ ID NO: 221), or SPGTGSPGTGSPGTGSPGTGSPGTGSPG (SEQ ID NO: 222).

[36] The molecule according to any one of [1] to

[35] above, which is a fusion polypeptide.

[37] The molecule according to any one of [1] to

[36] above, wherein the regulatory domain is N-terminal to the targeting domain.

[38] The molecule according to any one of [1] to

[36] above, wherein the regulatory domain is C-terminal to the targeting domain.

[39] The molecule according to any one of [2] to

[38] above, wherein the E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof comprises one or more domains selected from the group consisting of a RING (Really Interesting New Gene) domain, a U-box domain, a HECT (homologous to the carboxyl terminus of E6-AP) domain, and an RBR domain.

[40] The molecule according to any one of [1] to

[39] above, further comprising a detectable marker.

[41] 40. The molecule according to claim 40, wherein the detectable marker does not contain a lysine residue, and optionally the detectable marker is a hemagglutinin tag or a Glu-Glu epitope tag.

[42] The molecule according to any one of [1] to

[40] above, which is a protein having any one of the amino acid sequences of SEQ ID NOs: 156 to 167, 171 to 195, 202 to 204, 236 to 248, 253 to 256, 267, 270, and 272.

[43] The molecule according to any one of [1] to

[42] above, which comprises an intracellular localization signal such as a nuclear localization signal, a mitochondrial localization signal, or an endosomal localization signal.

[44] the molecule is capable of reducing the amount of the substrate by at least 20% compared to the amount of the substrate in the absence of the molecule; optionally, the molecule reduces the amount of the substrate in a cell by at least 20% compared to the amount of the substrate in an otherwise substantially identical cell that does not contain the molecule; The molecule according to any one of [1] to

[43] above.

[45] (i) a molecule according to any one of [1] to

[44] above; and (ii) a compound comprising a targeting moiety capable of targeting the molecule to a cell.

[46] 45. The compound according to claim 45, wherein the targeting moiety is a binding partner such as an antibody.

[47] The compound according to any one of claims 45 to 46, wherein the targeting moiety is a polypeptide fused to the molecule.

[48] Optionally, the polypeptide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 223 to 235, 249 to 252 and 259, 262 and 264. A polynucleotide encoding the molecule of any one of [1] to

[44] or the compound of

[47] .

[49] A vector comprising the polynucleotide described in

[48] , such as an adeno-associated virus (AAV) vector or a lentivirus vector.

[50] A host cell comprising the polynucleotide according to

[48] above or the vector according to

[49] above.

[51] A composition comprising the molecule according to any one of [1] to

[44] , the compound according to any one of

[45] to

[47] , the polynucleotide according to

[48] , the vector according to

[49] , or the host cell according to

[50] , and a further therapeutic agent.

[52] 51. The composition according to claim 51, wherein the additional therapeutic agent is an anti-cancer agent, an anti-viral agent, an anti-diabetic agent, an immunotherapeutic agent, an anti-inflammatory agent, an antibiotic, or any combination thereof.

[53] A molecule according to any one of [1] to

[44] , a compound according to any one of

[45] to

[47] , a polynucleotide according to

[48] , a vector according to

[49] , a host cell according to

[50] , or a composition according to

[51] or

[52] , for use in medicine.

[54] A pharmaceutical composition comprising the molecule according to any one of [1] to

[44] , the compound according to any one of

[45] to

[47] , the polynucleotide according to

[48] , the vector according to

[49] , the host cell according to

[50] , or the composition according to

[51] or

[52] , and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[55] Administering to an individual (i) a molecule according to any one of [1] to

[44] above and (ii) a compound comprising a targeting moiety capable of targeting the molecule to a cell; or Administering to the individual the polynucleotide according to

[48] or the vector according to

[49] , wherein the polynucleotide or vector encodes the molecule in the cell. A method for delivering the molecule according to any one of [1] to

[44] to the cells of the individual, comprising:

[56] the molecule is capable of reducing the amount of the substrate by at least 20% compared to the amount of the substrate in the absence of the molecule; optionally, the molecule reduces the amount of the substrate in a cell by at least 20% compared to the amount of the substrate in an otherwise substantially identical cell that does not contain the molecule; The method described in

[55] above.

[57] A compound for use in delivering the molecule according to any one of [1] to

[44] to a cell of an individual, comprising: (i) the molecule according to any one of [1] to

[44] ; and (ii) a targeting moiety capable of targeting the molecule to a cell.

[58] Use of a compound comprising (i) the molecule according to any one of [1] to

[44] above and (ii) a targeting moiety capable of targeting the molecule to a cell in the manufacture of a medicament for delivering the molecule according to any one of [1] to

[44] above to a cell of an individual.

[59] the molecule is capable of reducing the amount of the substrate by at least 20% compared to the amount of the substrate in the absence of the molecule; optionally, the molecule reduces the amount of the substrate in a cell by at least 20% compared to the amount of the substrate in an otherwise substantially identical cell that does not contain the molecule; The method described in

[58] above.

[60] (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence with at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; (b) a targeting domain capable of targeting the regulatory domain to the substrate; and A kit of parts comprising: Optionally, the kit does not include an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof. Kit of parts.

[61] The kit of parts according to

[60] , further comprising a linker capable of connecting the regulatory domain to the targeting domain.

[62] (a) a molecule according to any one of [1] to

[44] above; (b) a targeting moiety capable of targeting cells containing the substrate to be modulated; and A kit of parts comprising: Optionally, the targeting moiety is a binding partner, such as an antibody. Kit of parts.

[63] The kit of parts according to

[62] , further comprising a linker capable of connecting the regulatory domain to the targeting domain.

[64] (a) a polynucleotide encoding a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugating domain having an amino acid sequence having at least 80% sequence identity to a human E2 enzyme or a functional portion thereof; (b) a polynucleotide encoding a targeting domain capable of targeting the regulatory domain to a substrate; and A kit of parts comprising: Optionally, the kit does not include a polynucleotide encoding an E3 ubiquitin or ubiquitin-like ligase or a functional portion thereof. Kit of parts.

[65] The kit of parts according to

[64] , further comprising one or more promoter sequences capable of directing the expression of one or both of the polynucleotides in a cell containing the regulated substrate.

[66] (a) a polynucleotide encoding the molecule according to any one of [1] to

[44] above; and (b) a targeting moiety capable of targeting cells containing the substrate to be modulated; and Includes a kit of parts.

[67] The kit of parts according to any one of

[60] to

[66] , further comprising one or more reagents for evaluating the expression profile of cells containing the substrate to be regulated.

[68] The kit of parts according to any one of

[60] to

[67] above, further comprising a means for evaluating a property of the cell.

[69] The kit of parts according to any one of

[60] to

[68] , wherein the regulatory domain is as defined in any one of [1] to

[20] , and / or the targeting domain is as defined in any one of

[21] to

[26] .

[70] The kit of parts according to any one of

[60] to

[69] , wherein the substrate is as described in any one of

[25] to

[28] .

[71] A method for producing the molecule according to any one of [1] to

[44] above or the compound according to any one of

[45] to

[47] above, the method comprising expressing the polynucleotide according to

[48] above in a host cell.

[72] The method according to

[71] above, comprising introducing the polynucleotide according to

[48] above or the vector according to

[49] above into a host cell and expressing a polynucleotide encoding the molecule.

[73] A method for preventing or treating a disease or condition mediated by an abnormal level of a substrate or a form thereof in a subject, the method comprising administering to the subject the molecule described in any one of [1] to

[44] , the compound described in any one of

[45] to

[47] , the polynucleotide described in

[48] , the vector described in

[49] , the cell described in

[50] , the pharmaceutical composition described in

[54] , or the composition described in

[51] or

[52] .

[74] the molecule is capable of reducing the amount of the substrate by at least 20% compared to the amount of the substrate in the absence of the molecule; optionally, the molecule reduces the amount of the substrate in a cell by at least 20% compared to the amount of the substrate in an otherwise substantially identical cell that does not contain the molecule; The method described in

[73] above.

[75] A molecule according to any one of [1] to

[44] , a compound according to any one of

[45] to

[47] , a polynucleotide according to

[48] , a vector according to

[49] , a cell according to

[50] , a pharmaceutical composition according to

[54] , or a composition according to

[51] or

[52] , for use in preventing or treating a disease or condition mediated by an abnormal level of a substrate or a form thereof in a subject.

[76] Use of the molecule described in any one of [1] to

[44] , the compound described in any one of

[45] to

[47] , the polynucleotide described in

[48] , the vector described in

[49] , the cell described in

[50] , the pharmaceutical composition described in

[54] , or the composition described in

[51] or

[52] in the manufacture of a pharmaceutical for preventing or treating a disease or condition mediated by abnormal levels of a substrate or a form thereof in a subject.

[77] The method according to

[73] or

[74] or the use according to

[75] or

[76] , wherein the disease or condition is cancer, diabetes, an autoimmune disease, Alzheimer's disease, Parkinson's disease, pain, a viral disease, a bacterial disease, a prion disease, a fungal disease, a parasitic disease, arthritis, an immunodeficiency, or an inflammatory disease.

[78] A method for modulating a substrate, the method comprising contacting the substrate with the molecule according to any one of [1] to

[44] above under conditions effective for the molecule to modulate the substrate.

[79]

[78] The method of

[78] , wherein the modulating comprises degrading the substrate, or preventing the degradation of the substrate, or altering the intracellular location of the substrate, or modulating (e.g., increasing or decreasing) one or more activities of the substrate, or modulating the degree of post-translational modification of the substrate.

[80] 1. A method for identifying a substrate as a potential drug target, comprising: (a) providing a cell, tissue, or organ comprising said substrate; (b) contacting the cell, tissue, or organ with the molecule according to any one of [1] to

[44] , the compound according to any one of

[45] to

[47] , the polynucleotide according to

[48] , or the vector according to

[49] ; (c) assessing the effect of the molecule, compound, polynucleotide or vector on one or more properties of the cell, tissue or organ, wherein identification of an effect that correlates with a particular disease state indicates that the substrate is a potential drug target for the particular disease; A method comprising:

[81] 1. A method for assessing substrate function, comprising: (a) providing a cell, tissue, or organ comprising said substrate; (b) contacting the cell, tissue, or organ with the molecule according to any one of [1] to

[44] , the compound according to any one of

[45] to

[47] , the polynucleotide according to

[48] , or the vector according to

[49] ; (c) assessing the effect of the molecule, compound, polynucleotide, or vector on one or more properties of the cell, tissue, or organ; A method comprising:

[82] 1. A method for identifying a test agent that may be useful in preventing or treating a disease or condition mediated by abnormal levels of a substrate or form thereof, comprising: providing the substrate; providing a test agent comprising: (a) a regulatory domain comprising an E2 ubiquitin or ubiquitin-like conjugate domain having an amino acid sequence with at least 80% sequence identity to a human E2 ubiquitin or ubiquitin-like domain; and (b) a targeting domain capable of targeting the regulatory domain to a substrate, wherein optionally, the test agent does not comprise an E3 ubiquitin or ubiquitin-like ligase or portion thereof; contacting the substrate and the test agent under conditions effective for the test agent to facilitate modulation of the substrate; determining whether the test agent modulates the substrate; A method comprising:

[83] The method of

[82] , further comprising testing the test agent in an assay for the disease or condition.

[84] The method according to

[81] or

[82] , further comprising synthesizing, purifying and / or formulating the test agent.

[85] Use of the molecule according to any one of [1] to

[44] above or the compound according to any one of

[45] to

[47] above in drug target validation or drug discovery. [Example]

[0205] Example 1: Degradation of SHP2 protein in MDA-MB-231 cells comparing E3 and E2 fusion polypeptides. Introduction The goal of this experiment was to determine whether it was possible to produce biological PROTACs (referred to herein as fusion polypeptides) capable of target protein degradation by using an E2 ubiquitin-conjugating enzyme as a "regulatory" or "degradation" domain rather than the standard E3 ligase "degradation" domain. Previous studies have demonstrated the ability of biological PROTACs using E3 "degradation" domains to degrade target proteins (Portnoff et al., J. Biol. Chem., 2014 289(11):7844-5; Pan et al., Oncotarget, 2016 7(28):44299-44309; Fulcher et al., Open Biol, 2017 7(5).pii:170066). For this experiment, MDA-MB-231 breast cancer cells were transduced with lentiviral constructs encoding the fusion polypeptide and a control protein. The UBE2D1 E2 ubiquitin-conjugating enzyme was selected to be incorporated into the fusion protein as a linker and an N-terminal "degradation" domain upstream of the SHP2-binding monobody aCS3 (Sha et al., Proc Natl Acad Sci USA, 2013 110(37):14924-9). Controls included aCS3, the aCS3 monobody alone, VHL alone, UBE2D1 alone, and N- and C-terminal E3 ligase (VHL; von Hippel-Lindau) polypeptide fusions to untransduced control cells. The extent of targeted SHP2 degradation will be determined by Western blot analysis and densitometry of the Western blot bands.

[0206] Materials and Methods Lentiviral particles were generated as described in the "Lentiviral Particle Generation" section of the main "Methods" section. Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_aCS3 (SEQ ID NO: 149), HA_VHL (SEQ ID NO: 168), HA_VHL_Linker4_aCS3 (SEQ ID NO: 154), HA_aCS3_Linker4_VHL (SEQ ID NO: 200), HA_UBE2D1 (SEQ ID NO: 169), and HA_UBE2D1_Linker4_aCS3 (SEQ ID NO: 194).

[0207] MDA-MB-231 cells were transduced according to the method described in "Transduction of Cells with Lentiviruses" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. Lysates of untransduced MDA-MB-231 control ("cells") were also included. Western blot analysis of sample lysates was performed using rabbit anti-SHP2 (CST #3397; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution) and mouse anti-alpha tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution). Blots were then visualized on an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the SHP2 protein band was divided by the respective densitometry value of the loading control (alpha-tubulin). These values ​​were then expressed as a percentage of the SHP2 / α-tubulin value observed for control (untransduced) MDA-MB-231 cells.

[0208] result Figure 1A shows a Western blot using SHP2 protein and an alpha-tubulin loading control. Two replicate samples of HA_UBE2D1_Linker4_aCS3 (labeled "UBE2D1_aCS3" in the figure) show reduced SHP2 protein levels compared to the control sample. Densitometry quantification suggests a 90% reduction in SHP2 protein levels (Figure 1B). Samples expressing HA_VHL_Linker4_aCS3 (labeled "VHL_aCS3" in the figure) show no detectable SHP2, whereas the reverse-orientation HA_aCS3_Linker4_VHL sample (labeled "aCS3_VHL" in the figure) resulted in an approximately 70-80% reduction in SHP2 levels (Figure 1A and Figure 1B). HA_VHL alone (labeled "VHL" in the figure) and HA_UBE2D1 alone (labeled "UBE2D1" in the figure) controls did not appear to adversely affect SHP2 expression levels, although replicates of the HA_aCS3 monobody samples demonstrated some variability in SHP2 protein levels.

[0209] conclusion These data suggest that fusion polypeptides containing E2 ubiquitin-conjugating enzymes can reduce target protein expression. The most likely mechanism for this reduction is through targeted ubiquitination and subsequent proteasomal degradation. The data observed when testing VHL fusion constructs in different orientations suggest that the orientation of the binding and degradation domains relative to each other can affect the efficacy of targeted ubiquitination and, therefore, degradation by E3 ligases.

[0210] Example 2A: Investigation of the orientation of the fusion polypeptide domains and linker length in E3 ligase and E2 fusion polypeptides. Introduction The purpose of this experiment was to investigate the length of the linker between the "targeting" and "regulation / degradation" domains, as well as the orientation of these domains relative to each other, and to determine how these variables affected fusion polypeptide-mediated changes in target expression. The data shown in Figure 1 suggest that the N-terminal position of the VHL (E3 ligase) degradation domain resulted in greater target SHP2 degradation. This is the orientation reported by Fulcher et al. (Open Biol, 2017(5).pii:170066). For this experiment, MDA-MB-231 breast cancer cells and U20S osteosarcoma cells were transduced with lentiviral constructs encoding the fusion polypeptide and a control protein, and short (9-amino acid linker) and long (19-amino acid linker) versions were compared in both orientations. The UBE2D1 E2 ubiquitin-conjugating enzyme was selected as the E2 fusion polypeptide "regulation / degradation" domain, and VHL was selected as the E3 fusion polypeptide "degradation domain." The SHP2-binding monobody aCS3 (Sha et al., Proc Natl Acad Sci USA, 2013 110(37):14924-9) was used as the "binding" domain in all fusion polypeptide constructs. Controls included the aCS3 monobody alone, VHL alone, UBE2D1 alone, and untransduced control cells. The extent of targeted SHP2 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0211] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_aCS3 (SEQ ID NO: 149), HA_UBE2D1 (SEQ ID NO: 169), HA_UBE2D1_Linker2_aCS3 (SEQ ID NO: 159), HA_UBE2D1_Linker1_aCS3 (SEQ ID NO: 158), HA_aCS3_Linker2_UBE2D1 (SEQ ID NO: 203), HA_aCS3_Linker1_UBE2D1 (SEQ ID NO: 202), HA_VHL (SEQ ID NO: 168), HA_VHL_Linker2_aCS3 (SEQ ID NO: 153), HA_VHL_Linker1_aCS3 (SEQ ID NO: 152), HA_aCS3_Linker2_VHL (SEQ ID NO: 197), and HA_aCS3_Linker1_VHL (SEQ ID NO: 196).

[0212] MDA-MB-231 and U20S cells were transduced according to the method described in "Lentiviral Cell Transduction" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. Lysates of MDA-MB-231 and U20S untransduced controls ("cells") were also included. Western blot analysis of sample lysates was performed using rabbit anti-SHP2 (CST #3397; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution) and rabbit anti-GAPDH (CST #5174; 1:4,000) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution). Blots were visualized on an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the SHP2 protein band was divided by the respective densitometry value of the loading control (GAPDH). These values ​​were then expressed as a percentage of the SHP2 / GAPDH values ​​observed for control (untransduced) MDA-MB-231 and U20S cells, respectively.

[0213] result Figures 2A and 3A show Western blots with SHP2 protein and GAPDH loading control bands. The UBE2D1 "regulatory / degradation" domain construct is referred to by the shorter name E2D1 in Figures 2 and 3. In both MDA-MB-231 and U20S cell lines, the UBE2D1 (E2D1) fusion polypeptide construct resulted in a 60-90% reduction in SHP2 protein levels compared to control cells (Figures 2 and 3). In MDA-MB-231 cells, there was no significant variation in SHP2 protein reduction among different linker lengths and orientations (Figure 2). In U20S cells, there was slightly more variation, with the HA_aCS3_Linker1_UBE2D1 construct (labeled "aCS3_short_E2D1" in the figure) likely being the least effective format (Figure 3A). In both MDA-MB-231 and U20S cells, having the VHL E3 ligase degradation domain at the N-terminus resulted in the greatest reduction in SHP2 levels, regardless of linker length (Figures 2 and 3). Having the aCS3-binding domain N-terminal to the VHL degradation domain resulted in a less effective reduction in SHP2 protein levels in both cell lines (Figures 2 and 3).

[0214] conclusion These data suggest that fusion polypeptides containing E2 ubiquitin-conjugating enzymes may be less sensitive to domain orientation than E3 ligase fusion polypeptides. The data also demonstrated that E2 fusion polypeptides using UBE2D1 as the "regulatory / degradation" domain can reduce target SHP2 protein levels. Some variability was observed among some of the results, which may indicate differences in construct activity or construct quantity in each cell sample.

[0215] Example 2B: Further investigation of the length of the fusion polypeptide domain linker in E2 fusion polypeptides Introduction Following the study of the effect of orientation on PROTAC activity, the goal of this experiment was to further explore the length of the linker between the "targeting" and "regulation / degradation" domains to determine how varying linker lengths affected fusion polypeptide-mediated changes in target expression. The data shown in Figures 2A, 2B, 3A, and 3B demonstrate that fusion polypeptides containing an E2 ubiquitin-conjugating enzyme with either a 9-amino acid linker or a 19-amino acid linker were both able to reduce target SHP2 protein levels in MDA-MB-231 and U2OS cells. For this experiment, additional linkers were tested with lengths of 6, 11, 13, 16, 19, 23, 24, 26, and 28 amino acids. The UBE2D1 E2 ubiquitin-conjugating enzyme was again selected as the E2 fusion polypeptide "regulation / degradation" domain. The SHP2-binding monobody aCS3 (Sha et al., Proc Natl Acad Sci USA, 2013 110(37):14924-9) was again used as the "binding" domain in all fusion polypeptide constructs. Controls included untransduced control cells. The extent of targeted SHP2 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0216] Materials and Methods mRNA synthesis: A linear DNA template encoding the E2D1_aCS3_HA (UBE2D1_aCS3_HA) linker variant and consisting of a T7 promoter, 5' UTR, an open reading frame encoding the fusion polypeptide, a 3' UTR, and a poly(A) tail was used for in vitro transcription of mRNA as described elsewhere (Vaidyanathan S, et al., Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Mol Ther Nucleic Acids 12, 530-542 (2018)).

[0217] Fusion polypeptides were generated with the following configurations: UBE2D1_Linker_aCS3_HA, wild type UBE2D1 was used, the linker used corresponds to the following sequence:

[0218] [Table 1]

[0219] The fusion polypeptides used in these experiments correspond to the nucleic acid sequences of SEQ ID NOs: 223-235 which encode the amino acid sequences of SEQ ID NOs: 236-248.

[0220] Transfection of cells with mRNA: U20S cells were transfected with mRNA using RNAiMAX (Invitrogen) according to the manufacturer's instructions. 4 x 10 cells per well were transfected. 3 U2OS cells were aliquoted into collagen-coated 96-well plates and incubated at 37°C for 48 hours. The cells were then transfected with 100 ng of each mRNA encoding the fusion polypeptide (using RNAiMAX as a transfection reagent) per well and incubated at 37°C for 24 hours.

[0221] Quantitative SHP2 degradation in high-content imaging cells: Cells were then fixed using paraformaldehyde. SHP2 and HA tag levels were then probed using antibodies specific for these epitopes and detected using the Cytation5 High-content imaging system. SHP2 levels were normalized to a range of 0-100% based on the SHP2 levels found in untreated cells within each experiment. Data correspond to n = 3 or more biological replicates.

[0222] result Figure 3C shows the normalized fluorescence intensity of SHP2 protein for constructs containing linkers of various amino acid lengths. We investigated the dependence of the efficacy of the fusion polypeptide on the length of the linker between the UBE2D1 and aCS3-binding domains. In general, shorter linkers (6-20 amino acids in length) consistently showed higher SHP2 degradation activity, as indicated by a lower percentage of the normalized SHP2 signal, followed by longer linkers. However, even longer linkers (e.g., 24-28 amino acids in length) resulted in targeted degradation activity.

[0223] conclusion These data demonstrate that all linker lengths tested successfully induced targeted degradation. Furthermore, different linker compositions were tested, including linkers 19 amino acid residues long (linkers 2 and 11) and 28 amino acid residues long (linkers 15-18), demonstrating that altering the linker sequence does not abolish targeted degradation activity. All compositions tested resulted in targeted degradation. Therefore, regardless of linker length, targeted degradation activity can be maintained despite variations in linker sequence.

[0224] Example 3: Investigation of the effect of binding domain affinity on the activity of E3 ligase and E2 fusion polypeptides. Introduction The purpose of this experiment was to examine the activity of biological fusion polypeptides, measured by reduction of target protein levels, using the aCS3 monobody or the mutant aCS3 V33R as the binding domain. The fusion polypeptide variants were tested in MDA-MB-231 and U20S cells. The standard aCS3 monobody, with its reported high affinity for SHP2 (SHP2 C-SH2 domain Kd = 4-9.1 nM), was compared with the V33R aCS3 mutant (SHP2 C-SH2 domain Kd = 1.2 μM), with its lower affinity (Sha et al., Proc Natl Acad Sci USA, 2013 110(37):14924-9 and supplementary information). In the Sha et al. publication, the aCS3 monobody was referred to as CS3. Controls included the aCS3 monobody alone, VHL alone, UBE2D1 alone, and untransduced control cells. Fusion polypeptides containing the N- and C-terminal regulatory / degradation domains of UBE2D1 or VHL were tested with either the standard aCS3-binding domain or the aCS3 V33R-binding domain. All fusion polypeptide constructs tested had a 19-amino acid "long" linker. The extent of targeted SHP2 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0225] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_aCS3 (SEQ ID NO: 149), HA_UBE2D1 (SEQ ID NO: 169), HA_UBE2D1_Linker2_aCS3 (SEQ ID NO: 159), HA_UBE2D1_Linker2_aCS3(V33R) (SEQ ID NO: 160), HA_aCS3_Linker2_UBE2D1 (SEQ ID NO: 161), No. 203), HA_aCS3(V33R)_Linker2_UBE2D1 (SEQ ID NO: 195), HA_VHL (SEQ ID NO: 168), HA_VHL_Linker2_aCS3 (SEQ ID NO: 153), HA_VHL_Linker2_aCS3(V33R) (SEQ ID NO: 155), HA_aCS3_Linker2_VHL (SEQ ID NO: 197), and HA_aCS3(V33R)_Linker2_VHL (SEQ ID NO: 201).

[0226] MDA-MB-231 and U20S cells were transduced according to the method described in "Lentiviral Cell Transduction" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. Lysates from untransduced MDA-MB-231 and U20S controls ("cells") were also included. Western blot analysis of sample lysates was performed using rabbit anti-SHP2 (CST #3397; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution) and rabbit anti-GAPDH (CST #5174; 1:4,000) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution). Blots were then visualized on an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the SHP2 protein band was divided by the respective densitometry value of the loading control (GAPDH), and these values ​​were then presented as a percentage of the SHP2 / GAPDH values ​​observed for control (untransduced) MDA-MB-231 and U20S cells, respectively.

[0227] result Figures 4A and 5A show Western blots with SHP2 protein and GAPDH loading control bands. The UBE2D1 "regulatory / degradation" domain construct uses the shorter name "E2D1" in Figures 4 and 5. In both MDA-MB-231 and U20S, all samples with the standard aCS3-binding domain showed a greater reduction in SHP2 protein levels than the mutated low-affinity variant aCS3(V33R) (Figures 4 and 5). In MDA-MB-231 cells, UBE2D1(E2D1) fusion polypeptide constructs with the standard aCS3-binding domain (both orientations) showed approximately 80-90% reduction in SHP2 protein (Figure 4B). In comparison, UBE2D1(E2D1) fusion polypeptide constructs with the mutated aCS3(V33R)-binding domain (both orientations) showed approximately 35-60% reduction in SHP2 protein (Figure 4B). These differences were even greater when we examined the N-terminal VHL E3 ligase fusion polypeptide, in which SHP2 protein was undetectable with aCS3. However, the aCS3(V33R) variant reduced SHP2 protein by 40% compared to control cells (Figure 4B). In U2OS cells (Figure 5), a similar pattern of results to that described for MDA-MB-231 cells (Figure 4) was observed.

[0228] conclusion These data suggest that by decreasing the binding affinity of the binding domain, the activity of the fusion polypeptide is reduced, leaving more of the target protein undegraded in the cell. These data suggest that increasing the affinity of the binding domain can increase the amount of target protein degradation. Again, the data indicate that the N-terminal VHL E3 ligase "degradation" domain was the most active orientation for the E3 ligase fusion polypeptide tested. The E2 ubiquitin-binding "regulatory / degradation" domain constructs using UBE2D1 and aCS3 demonstrate fairly comparable activity in both orientations tested so far. Some variability was observed between experiments, which may be due to differences in transduction efficiency and lentiviral titer.

[0229] Example 4: Degradation of endogenous KRas protein using E2 fusion polypeptides. Introduction The purpose of this experiment was to determine whether fusion polypeptides containing an E2 ubiquitin-conjugating enzyme as a "regulatory / degradation" domain could be used to degrade alternative endogenous target proteins. This was tested in two different cell lines, MDA-MB-231 and Ad293 cells. The binding domain of the fusion polypeptide constructs tested was either the designed ankyrin repeat protein (DARPin) K19 or E3_5. K19 binds both GTP- and GDP-bound KRas (Bery et al., Nat Commun. 2019 10(1):2607). E3_5 served as a negative control, nonselective DARPin (Binz et al., J Mol Biol, 2003 332(2):489-503). Controls included DARPin E3_5 alone, VHL alone, and untransduced control cells. All fusion polypeptides contained the N-terminal "binding domain" of DARPin K19 or E3_5 and the C-terminal "regulatory / degradation" domain of UBE2D1 or VHL. The domains in the constructs were linked by a 20-amino acid linker ("Linker 3"). The extent of targeted KRas degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0230] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_VHL (SEQ ID NO: 168), HA_E3_5 (SEQ ID NO: 151), HA_K19_Linker3_VHL (SEQ ID NO: 198), HA_E3_5_Linker3_VHL (SEQ ID NO: 199), HA_K19_Linker3_UBE2D1 (SEQ ID NO: 204), and HA_E3_5_Linker3_UBE2D1 (SEQ ID NO: 205).

[0231] MDA-MB-231 and Ad293 cells were transduced according to the method described in "Transduction of Cells with Lentiviruses" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. MDA-MB-231 and Ad293 non-transduced control ("cell") lysates were also included. Western blot analysis of sample lysates was performed using mouse anti-KRas (LS-Bioscience #LS-C175665; 1:2000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution) and mouse anti-alpha tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution). The blots were then visualized using Odyssey, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the KRas protein band was divided by the densitometry value of the loading control (alpha-tubulin). These values ​​were then expressed as a percentage of the KRas / α-tubulin values ​​observed for control (untransduced) MDA-MB-231 and Ad293 cells, respectively.

[0232] result In the Western blots shown in Figure 6, greater than 80% degradation of endogenous KRas was observed in both MDA-MB-231 and Ad293 cells using the E2 ubiquitin-conjugating enzyme fusion polypeptide K19_E2D1 (HA_K19_Linker3_UBE2D1). The UBE2D1 "regulatory / degradation" domain construct is referred to as "E2D1" in Figure 6. The negative control fusion polypeptide E3_5_E2D1 (HA_E3_5_Linker3_UBE2D1) did not result in any KRas degradation in these cells (Figures 6A and 6B). In this format, the E2 fusion polypeptide (using UBE2D1) was more effective than the E3 ligase fusion polypeptide (using VHL) in reducing KRas protein levels in both MDA-MB-231 and Ad293 cells. The K19_VHLE3 ligase fusion polypeptide only showed a reduction in KRas protein levels in MDA-MB-231 cells, but not in Ad293 cells.

[0233] conclusion These data demonstrate that E2 ubiquitin-conjugating enzyme "regulatory / degradation" domains can regulate targets other than SHP2. In this case, the binding domain (DARPinK19) recruited endogenous KRas, resulting in a downstream reduction in KRas protein levels. In the linker and domain orientations tested, KRas-targeting E2 fusion polypeptides were able to demonstrate activity in both MDA-MB-231 and Ad293 cells. Conversely, KRas-targeting E3 fusion polypeptides had some activity in MDA-MB-231 cells but no activity in Ad293 cells.

[0234] These data suggest either (i) that the tested format is suboptimal for E3 fusion polypeptide activity (this orientation was previously less effective for SHP2-targeted VHL fusion polypeptides, but protein levels were consistently detected despite this reduction in SHP2), and / or (ii) that E3 ligase fusion polypeptides may be subject to variable activity depending on the cellular background, e.g., due to the expression levels of specific adaptor proteins required for the EloB / C / CUL2 / RBX1 E3 ligase machinery (see Figure 7). This is clearly an advantage of using E2 fusion polypeptides, as they are less dependent on the expression of multiple endogenous proteins to effect target binding and ubiquitin transfer, potentially allowing activity in a larger panel of cell types.

[0235] Example 5A: Investigation of a panel of core E2 ubiquitin and ubiquitin-like conjugating enzymes as "regulatory / degradation" domains in SHP2-targeting fusion polypeptides. Introduction The goal of this experiment was to determine which core E2 ubiquitin or ubiquitin-like conjugating enzyme sequence, when expressed in an E2 fusion polypeptide format, i.e., coreE2_Linker2_aCS3, could maximally reduce target protein expression. Twenty-six different core E2 ubiquitin or ubiquitin-like conjugating enzyme sequences were tested, and expression of the fusion polypeptide constructs was examined. The resulting SHP2 protein levels were determined by Western blot and compared to the E2D1_aCS3 fusion polypeptide used in the previous example. The panel of constructs was tested in MDA-MB-231 and U20S cells. The core E2 domains tested were UBE2D1, UBE2B, UBE2C, UBE2D2, UBE2D3, UBE2E1, UBE2F, UBE2G1, UBE2G2, UBE2H, UBE2I, UBE2J2, UBE2K, UBE2L3, UBEL6, UBE2M, UBE2O, UBE2Q1, UBE2Q2, UBE2R1, UBE2S, UBE2T, UBE2U, UBE2W, BIRC6, and UFC1. In Western blots, these samples were designated by the shortened nomenclature lacking the initial letters "UB," such that UBE2D1 was designated as E2D1 (Figures 8A and 9A). Controls included the aCS3 monobody alone and untransduced control cells. The extent of targeted SHP2 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0236] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_aCS3 (SEQ ID NO: 149), HA_UBE2D1_Linker2_aCS3 (SEQ ID NO: 159), HA_UBE2B_Linker2_aCS3 (SEQ ID NO: 156), HA_UBE2C_Linker2_aCS3 (SEQ ID NO: 157), HA_UBE2D2_Linker2_aCS3 (SEQ ID NO: 171), HA_UBE2D3_Linker2_aCS3 ( SEQ ID NO: 172), HA_UBE2E1_Linker2_aCS3 (SEQ ID NO: 173), HA_UBE2F_Linker2_aCS3 (SEQ ID NO: 174), HA_UBE2G1_Linker2_aCS3 (SEQ ID NO: 175), HA_UBE2G2_Linker2_aCS3 (SEQ ID NO: 176), HA_UBE2H_Linker2_aCS3 (SEQ ID NO: 177), HA_UBE2I_Linker2_aCS3 (SEQ ID NO: 178), HA_UBE2J2_Linker2_aCS3 (SEQ ID NO: 179), No. 179), HA_UBE2K_Linker2_aCS3 (SEQ ID NO: 180), HA_UBE2L3_Linker2_aCS3 (SEQ ID NO: 181), HA_UBEL6_Linker2_aCS3 (SEQ ID NO: 182), HA_UBE2M_Linker2_aCS3 (SEQ ID NO: 183), HA_UBE2O_Linker2_aCS3 (SEQ ID NO: 184), HA_UBE2Q1_Linker2_aCS3 (SEQ ID NO: 185), HA_UBE2Q2_Linker2_aCS3 (SEQ ID NO: 1 86), HA_UBE2R1_Linker2_aCS3 (SEQ ID NO: 187), HA_UBE2S_Linker2_aCS3 (SEQ ID NO: 188), HA_UBE2T_Linker2_aCS3 (SEQ ID NO: 189), HA_UBE2U_Linker2_aCS3 (SEQ ID NO: 190), HA_UBE2W_Linker2_aCS3 (SEQ ID NO: 191), HA_BIRC6_Linker2_aCS3 (SEQ ID NO: 192), and HA_UFC1_Linker2_aCS3 (SEQ ID NO: 193).

[0237] MDA-MB-231 and U20S cells were transduced according to the method described in "Transduction of cells with lentiviruses" and prepared for Western blot analysis as described in the Main Methods section under "Western blot analysis and quantification." MDA-MB-231 and U20S untransduced control ("cell") lysates were also included. Western blot analysis of sample lysates was performed using mouse anti-SHP2 (Abcam #ab76285; 1:1000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution), mouse anti-α-tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution), and rabbit anti-HA tag (Abcam #ab137838; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution). Blots were then visualized using an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the SHP2 protein band was divided by the respective densitometry value of the loading control (alpha-tubulin). These values ​​are presented as a percentage of the SHP2 / alpha-tubulin value observed for the UBE2D1_aCS3E2 fusion polypeptide (Figures 8B and 9B) to determine whether any other core E2s may have a greater effect on target protein levels in MDA-MB-231 and U20S cells, respectively.

[0238] result Consistent with previous data, degradation of SHP2 protein was observed using HA_UBE2D1_Linker2_aCS3 (labeled "E2D1_aCS3" in the figures) in both MDA-MB-231 and U20S cells (Figures 8 and 9, respectively). Several of the core E2 constructs tested in this format (N-terminal E2 core domain and C-terminal aCS3-binding domain) resulted in a small decrease in SHP2 protein levels as determined by Western blot (Figures 8A and 9A). Many of the constructs tested did not appear to reduce SHP2 protein levels. Consistently, in both MDA-MB-231 and U20S cells, core HA_UBE2B_Linker2_aCS3 (labeled "E2B_aCS3" in the figures) and, to a lesser extent, HA_UBE2D2_Linker2_aCS3 (labeled "E2D2_aCS3" in the figures) reduced SHP2 protein levels to a greater extent than HA_UBED1_Linker2_aCS3, as quantified by Western blot band densitometry and normalized to loading controls (Figures 8B and 9B). HA Western blots demonstrate the relative expression levels and / or stability of these HA-tagged fusion polypeptide constructs. Both HA_UBE2D1_Linker2_aCS3 and HA_UBE2D2_Linker2_aCS3 showed minimal HA bands, indicating poor construct expression or poor stability of the expressed constructs in the cells. On the other hand, in both cell lines, the HA_UBE2B_Linker2_aCS3 construct showed higher expression levels of HA-tagged proteins than HA_UBE2D1_Linker2_aCS3 and HA_UBE2D2_Linker2_aCS3 (Figures 8A and 9A). Most of the tested constructs with diverse sequences of the core E2 "regulatory / degradation" domain showed high HA band intensities by Western blot, indicating high levels of construct expression and / or stability in cells.

[0239] conclusion These data indicate that target modulation resulting in a reduction in cellular expression levels of the target protein using the HA_UBE2B_Linker2_aCS3, HA_UBE2D1_Linker2_aCS3, and HA_UBE2D2_Linker2_aCS3 constructs resulted in the lowest SHP2 protein levels in both MDA-MB-231 cells. Many of the other core E2 constructs only minimally reduced target expression, if at all. In the case of E2-ubiquitin-conjugating enzyme core domain fusion polypeptides, target degradation is likely via a ubiquitin-mediated mechanism involving target ubiquitination, polyubiquitination, and ultimately proteasomal degradation. The E2 ubiquitin-like conjugating enzyme core fusion polypeptides (e.g., HA_UBE2F_Linker2_aCS3, HA_UBE2I_Linker2_aCS3, and HA_UBE2M_Linker2_aCS3) may post-translationally modify or regulate target proteins in other ways, such as by transfer of ubiquitin-like molecules in the absence of ubiquitin transfer itself.

[0240] Example 5B: Comparison of core E2 ubiquitin and ubiquitin-like conjugating enzymes as "regulatory / degradation" domains in K19-targeting fusion polypeptides. Introduction Following the determination that UBE2D1 (E2D1) functions in either orientation for the degradation of different endogenous target proteins (e.g., SHP2 and K19), the purpose of this experiment was to determine whether a different core E2 enzyme, UBE2B, could also reduce target protein expression regardless of orientation. Constructs were tested in U20S cells. The core E2 domains tested were UBE2D1 (as a positive control) and UBE2B. In Western blots, these samples were designated by the shortened nomenclature, lacking the initial letters "UB," so that UBE2D1 is shown as E2D1 (Figure 13). Controls included untransduced control cells. The extent of target K19 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0241] Materials and Methods Lentiviral particles encoding the following fusion polypeptides were produced in HEK293FT cells: HA_K19_Linker2_UBE2D1 (SEQ ID NO: 253), HA_UBE2D1_Linker2_K19 (SEQ ID NO: 254), HA_K19_Linker2_UBE2B (SEQ ID NO: 255), and HA_UBE2B_Linker2_K19 (SEQ ID NO: 256). PROTACs containing the following degradation domains were investigated: UBE2D1 (E2D1), UBE2B (E2B), and VHL. KRas-targeting PROTACs were tested in both the "binding domain_degradation domain" and "degradation domain_binding domain" orientations. The negative control DARPinE3_5 was used as a negative control binding domain in combination with various degradation domains in both orientations (SEQ ID NOs: 274-276 and 278). Fusion polypeptides with the E3 degradation domain were also included as controls: HA_VHL_Linker2_K19 (SEQ ID NO: 277), and HA_K19_Linker2_VHL (SEQ ID NO: 279).

[0242] HPAC pancreatic cancer cells were transduced according to the method described in "Transduction of Cells with Lentiviruses" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. Lysates from untransduced HPAC control ("cells") were also included. Western blot analysis of sample lysates was performed using mouse anti-KRas (LS-Bioscience #LS-C175665; 1:2000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution) and mouse anti-alpha tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution). Blots were then visualized using Odyssey, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the KRas protein band was divided by the respective densitometry value of the loading control (alpha-tubulin). These values ​​were then expressed as a percentage of the KRas / α-tubulin value observed for control (untransduced) HPAC cells.

[0243] result Figure 13 shows a Western blot with K19 protein and α-tubulin loading control bands. The UBE2D1 and UBE2B' "regulatory / degradation" domain constructs use the shorter names E2D1 and E2B in Figure 13. In HPAC cells, both orientations of the UBE2D1 (E2D1) fusion polypeptide construct resulted in reduced K19 protein levels compared to the control (Figure 13A). Similarly, both orientations of the VHL fusion polypeptide construct resulted in reduced K19 protein levels compared to the control, although the K19_VHL orientation showed a lower level of reduction. These data correlate with data seen in other cell lines, demonstrating that HPAC pancreatic cancer cells are an additional valid model for investigating PROTAC activity. Referring to Figure 13B, UBE2B (E2B) fusion polypeptide constructs (both orientations) were compared to UBE2D1 (E2D1) fusion polypeptide constructs (both orientations), all of which resulted in a 70-90% reduction in K19 protein levels (K19_E2D1 87% reduction; E2D1_K19 86% reduction; K19_E2B 85% reduction; and E2B_K19 79% reduction).

[0244] conclusion These data indicate that use of the UBE2B degradation domain can result in degradation of KRas protein expression, as well as SHP2 expression in the alternative construct of Example 5A. Furthermore, both orientations of the PROTAC fusion polypeptide can result in targeted degradation. Collectively, these data demonstrate that multiple E2 ubiquitin or ubiquitin-like conjugate domains fused to multiple targeting domains result in functional PROTACs, regardless of the orientation of these domains in the fusion polypeptide.

[0245] Example 6A: Lysine residues in the aCS3 binding domain are mutated to determine whether this improves the activity and stability of the fusion polypeptide in cells. Introduction The purpose of this experiment was to determine whether the three lysine residues present in the aCS3 monobody (K7, K55, and K64) "binding" domain within the fusion polypeptide are prone to self-ubiquitination. If these lysine residues are ubiquitinated, this could result in fusion polypeptide degradation, reduced stability, and reduced activity in cells. The lysine residues were mutated individually and in combination as part of the UBE2D1_aCS3 construct. Structural modeling indicated which amino acid residue changes should maintain monobody stability. Lysine residue K7 was mutated to glutamine (K7Q). Lysine residue K55 was mutated to tyrosine (K55Y), and lysine residue K64 was mutated to histidine (K64H). The effects on SHP2 degradation and fusion polypeptide expression in U2OS cells expressing fusion polypeptides containing these aCS3 variants were measured by Western blot probing for SHP2 protein and HA tag expression levels, respectively. Alpha-tubulin expression levels were determined by Western blot as a loading control. Control samples included the aCS3 monobody alone, UBE2D1_aCS3(WT), and untransduced control cells. The extent of targeted SHP2 degradation was determined by Western blot analysis and quantified by densitometry of the Western blot bands.

[0246] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_aCS3 (SEQ ID NO: 149), UBE2D1_Linker2_aCS3 (SEQ ID NO: 159), UBE2D1_Linker2_aCS3(K7Q) (SEQ ID NO: 161), UBE2D1_Linker2_aCS3(K55Y) (SEQ ID NO: 162), UBE2D1_Linker 2_aCS3(K64H) (sequence number 163), UBE2D1_Linker2_aCS3(K7Q, K55Y) (sequence number 164), UBE2D1_Linker2_aCS3(K7Q, K64H) (sequence number 165), UBE2D1_Linker2_aCS3(K55Y, K64H) (sequence number 166), and UBE2D1_Linker2_aCS3(K7Q, K55Y, K64H) (sequence number 167).

[0247] U20S cells were transduced according to the method described in "Transduction of cells with lentiviruses" and prepared for Western blot analysis as described in the Main Methods section under "Western blot analysis and quantification." U20S untransduced control ("cells") lysates were also included. Western blot analysis of sample lysates was performed using mouse anti-SHP2 (Abcam #ab76285; 1:1000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution), mouse anti-α-tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution), and rabbit anti-HA tag (Abcam #ab137838; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution). Blots were then visualized using an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the SHP2 protein band was divided by the respective densitometry value of the loading control (alpha tubulin). These values ​​are presented as a percentage of the SHP2 / alpha tubulin value observed for U20S control cells. Additionally, for each sample, the densitometry value of the HA-tagged protein band was divided by the respective densitometry value of the loading control (alpha tubulin). These values ​​are presented as a percentage of the HA / alpha tubulin value observed for UBE2D1_aCS3(WT) to determine whether fusion polypeptide protein expression in cells could be improved by removing the binding domain lysine residues.

[0248] result These results indicate that all HA_UBE2D1_Linker2_aCS3 (labeled "E2D1_aCS3" in the figure) samples (wild-type and lysine mutants) were able to induce at least a 75% reduction in SHP2 protein expression (Figures 10A and 10B). These results appear to be comparable across all variants tested. All lysine mutant variants also showed increased HA expression levels compared to the wild-type aCS3 variant (Figure 10C). The greatest increase in HA expression appeared to involve the mutation of lysine at position 7 (K7Q), either alone or in combination with other lysine mutations, with the highest levels observed for the triple mutant (K7Q, K55Y, K64H; Figure 10C).

[0249] conclusion These data indicate that removing lysine residues from the aCS3 monobody sequence appeared to increase the level of fusion polypeptide expression in cells without adversely affecting the extent of targeted SHP2 degradation in cells. The key residue that appeared to increase HA expression while maintaining the fusion polypeptide's ability to interact with target SHP2 was K7. All variants containing this mutation appeared to exhibit improved stability and activity profiles compared to HA_UBE2D1_Linker2_aCS3WT. The triple mutant (K7Q, K55Y, K64H) showed the greatest increase in HA expression and target degradation. These data suggest that within the E2 fusion polypeptide construct, lysine residues represent a potential for self-ubiquitination that can be resolved by replacing them with alternative residues. In this case, we performed in-house structural modeling studies to select the best mutations for maintaining aCS3 monobody stability. These data indicate that activity appears to be maintained, as targeted SHP2 degradation was either equivalent or increased in the variants tested.

[0250] Example 6B: Mutating the catalytic site of the UBE2D1 or UBE2B regulatory domain of the fusion polypeptide or reducing the binding domain affinity for the target protein reduces the degradation of the target protein. Introduction The purpose of this experiment was to further examine the ability of point mutations to alter the activity of the fusion polypeptide. These experiments aimed to determine whether three lysine residues present in the "binding" domain of the aCS3 monobody (K7, K55, and K64) within the fusion polypeptide are prone to self-ubiquitination in UBE2D1 and UBE2B, which have different cysteine ​​catalytic sites. These mutants were also tested using the V33R mutation of aCS3 to reduce the affinity of the binding domain for the target protein SHP2. Finally, UBE2D1 was further mutated at residue F62, which is involved in interactions with several E3 ligases, to determine its effect on activity.

[0251] Materials and Methods mRNA synthesis: Linear DNA templates encoding SHP2-targeting fusion polypeptides with various point mutations in the binding and regulatory domains, consisting of a T7 promoter, a 5' UTR, an open reading frame encoding the fusion polypeptide, a 3' UTR, and a poly(A) tail, were used for in vitro transcription of mRNA as described elsewhere (Vaidyanathan S, et al., Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Mol Ther Nucleic Acids 12, 530-542 (2018)).

[0252] The mRNA sequences used encode the following fusion polypeptides: UBE2D1_Linker2_aCS3(K7Q,K55Y,K64H)_HA (SEQ ID NO: 240), UBE2D1(C85A)_Linker2_aCS3(K7Q,K55Y,K64H)_HA (SEQ ID NO: 266), UBE2D1_Linker2_aCS3(K7Q,K55Y,K64H,V33R)_HA (SEQ ID NO: 267), UBE2D1(C85A)_Linker2_aCS3(K7Q,K55Y,K64H,V33R)_HA (SEQ ID NO: 268), UBE2D1(F62A)_Linker2_aCS3 (K7Q,K55Y,K64H)_HA (sequence number 269), UBE2B_Linker2_aCS3(K7Q,K55Y,K64H)_HA (sequence number 270), UBE2B(C88A)_Linker2_aCS3(K7Q,K55Y,K64H)_HA (sequence number 271), UBE2B_Linker2_aCS3(K7Q,K55Y,K64H,V33R)_HA (sequence number 272), and UBE2B(C88A)_Linker2_aCS3(K7Q,K55Y,K64H,V33R)_HA (sequence number 273).

[0253] Transfection of cells with mRNA: U20S cells were transfected with mRNA using RNAiMAX (Invitrogen) according to the manufacturer's instructions. 3.5 x 10 cells per well were transfected. 5 U2OS cells were seeded into 6-well plates and incubated at 37°C for 24 hours. The cells were then transfected with 3 μg of each mRNA-encoding fusion polypeptide per well (using RNAiMAX as the transfection reagent) and incubated at 37°C for 24 hours.

[0254] Western blot analysis and quantification: The medium was removed from the cells and washed with PBS. Cells were harvested using Accutase (Sigma) and incubated at 37°C for 3 minutes. The Accutase was then neutralized by the addition of complete medium. The cell suspension was then collected and centrifuged at 1200 rpm (300 x g) for 5 minutes to pellet the cells. The cell pellet was washed with PBS and transferred to a 1.5 mL Eppendorf tube. These tubes were then centrifuged at 1200 rpm (300 x g) for 5 minutes, and the supernatant was discarded. The cell pellet was lysed in RIPA lysis buffer (Thermo Fisher Scientific) containing a 1:100 dilution of a protease and phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was incubated on ice for 30 minutes and then clarified by centrifugation at 15,000 rpm (17,000 x g) for 10 minutes at 4°C. The protein concentration of each lysate was determined by BCA assay (Pierce / Thermo Fisher Scientific, according to the manufacturer's instructions). Then, 40 μg of lysate from each cell line was loaded per well onto a 4-12% BOLT gel (Thermo Fisher Scientific), run at 200 V for 25 minutes, and then transferred to a membrane using iBlot according to the manufacturer's instructions (Thermo Fisher Scientific). The membrane was then blocked in Odyssey blocking buffer (Li-cor) and subjected to Western blot analysis using the appropriate antibodies (see Table 2, below).

[0255] Blots were then visualized on an Odyssey system according to the manufacturer's instructions (Li-cor), and densitometry of Western blot bands was measured using Image Studio software according to the manufacturer's instructions (Li-cor).

[0256] result To further explore the ability of point mutations to alter fusion polypeptide activity, a panel of mutant binding domain and regulatory domain fusion polypeptides was examined. U20S cells were transfected with mRNA encoding a panel of variant fusion polypeptides that target the SHP2 protein for ubiquitination and subsequent degradation. Cells were transfected using RNAiMAX, incubated for 24 hours, harvested, and SHP2 protein levels analyzed by Western blot. The binding domains of the fusion polypeptide variants used in this example contained K7Q, K55Y, and K64H point mutations to increase fusion polypeptide expression compared to the unmutated aCS3 binding domain (as shown in Figure 10C).

[0257] Additional point mutations investigated included: (i) Mutating the catalytic cysteine ​​residues in the regulatory domains, e.g., UBE2D1 (C85A) and UBE2B (C88A), resulted in the rescue of SHP2 protein levels to normal (or near-normal) levels by inactivating the catalytic residues in the regulatory domains (see Figures 10D, 10E, and 10F); (ii) Inclusion of an additional V33R mutation in aCS3 reduced the affinity of the binding domain for the target protein SHP2, thereby bringing SHP2 protein levels closer to normal expression levels. SHP2 expression levels were not fully rescued. This is likely because the V33R point mutation in aCS3 did not completely abolish target binding, but instead reduced affinity by approximately 100-fold (Sha et al., Proc. Natl. Acad. Sci. USA, 2013 110(37):14924-9, and supplementary information; see Figures 10D, 10E, and 10F); and (iii) to determine the effect on activity of mutating UBE2D1 residue F62, which is involved in the interaction with E3 ligase, resulting in complete rescue of SHP2 protein (i.e., F62A);

[0258] conclusion These data indicate that mutating the catalytic cysteine ​​of an E2 regulatory domain (e.g., UBE2D1 or UBE2B) to alanine inactivates the regulatory domain and inhibits target protein modification (in this case, ubiquitination and degradation of SHP2). Furthermore, reducing the affinity of the binding domain for the target protein also reduces the extent of target protein modification. In this example, the V33R mutation in aCS3 reduced its binding affinity for SHP2 by approximately 100-fold (Sha et al., Proc. Natl. Acad. Sci. US Patent Application Publication No. 2013110(37):14924-9 and supplementary information). Finally, these data suggest that interaction between E3 ligases and E2 regulatory domains may be important for regulatory domain activity, as the F62A mutation in UBE2D1 appears to abolish SHP2 degradation. Structural studies have shown that residue F62 is involved in the interaction between UBE2D1 and the E3 ligase RNF4 (Gundogdu and Walden, Protein Science. 2019;28:1758-1770), which may be involved in catalyzing ubiquitination. Mutation of this residue to alanine was hypothesized to disrupt the E3 interaction with UBE2D1, which would prevent the fusion polypeptide from targeting SHP2 for degradation.

[0259] Example 7: Nuclear target degradation using E2 ubiquitin-conjugating enzyme fusion polypeptides. Introduction The goal of this experiment was to determine whether the E2 fusion polypeptide format could successfully degrade a primarily nuclear target. Human Antigen R was the target chosen because a single-domain antibody / nanobody sequence was available for this target. Human Antigen R (HuR / ELAVL1) is an RNA-binding protein involved in the stabilization and translational upregulation of target mRNAs. HuR is primarily localized in the nucleus but, in response to different stimuli, is transported to the cytoplasm, a process regulated by several post-translational modifications, which also affect binding to target mRNAs (Doller et al., Cell Signal., 2008 20:2165-2173). Two distinct nanobody sequences were selected for this experiment: HuR8 and HuR17. The binding affinity of HuR8 for the target Human Antigen R is 2100 nM, and that of HuR17 is 30 nM. A control Cas9V targeting the Cas9 protein was also chosen. HH The nanobody binding domain was included. Cas9 is a bacterial protein and is not endogenously expressed in mammalian cells. Therefore, Cas9V HH Nanobodies should not selectively bind to any protein in mammalian cells. HH Nanobodies were cloned into UBE2D1 fusions in both orientations: (UB)E2D1_Linker_V HH and V HH _Linker_(UB)E2D1. The linker used was the 19 amino acid linker 2 (SEQ ID NO: 142). Lentiviral particles encoding these constructs were transduced into two different cell lines (MDA-MB-231 and U20S), and the resulting effect on HuR expression was examined by Western blot analysis.

[0260] Materials and Methods Lentiviral particles encoding the following fusion polypeptides (or individual components) were produced in HEK293FT cells: HA_UBE2D1_Linker2_Cas9, HA_UBE2D1_Linker2_HuR8, HA_UBE2D1_Linker2_HuR17, HA_Cas9_Linker2_UBE2D1, HA_HuR8_Linker2_UBE2D1, and HA_HuR17_Linker2_UBE2D1.

[0261] MDA-MB-231 and U20S cells were transduced according to the method described in "Transduction of Cells with Lentiviruses" and prepared for Western blot analysis as described in the "Western Blot Analysis and Quantification" section of the Main Methods section. Western blot analysis of sample lysates was performed using rabbit anti-Hur / ELAVL1 (CST #12582; 1:1000 dilution) with secondary goat anti-rabbit IRDye800 (Licor #925-32211; 1:15,000 dilution) and mouse anti-alpha tubulin (Licor #926-42213; 1:10,000 dilution) with secondary goat anti-mouse IRDye680RD (Licor #926-68070; 1:15,000 dilution). Blots were then visualized using an Odyssey system, and Western blot densitometry was performed using Image Studio software. For each sample, the densitometry value of the HuR protein band was divided by the respective densitometry value of the loading control (alpha-tubulin). These values ​​are then presented as a percentage of the HuR / alpha-tubulin value observed for the respective control lysate (e.g., HA_Cas9_Linker2_UBE2D1 or HA_UBE2D1_Linker2_Cas9, depending on the orientation of the fusion protein tested).

[0262] result These results show that HuR protein expression was reduced in MDA-MD-231 and U20S cell lines expressing the HA_UBE2D1_Linker2_HuR17 (labeled "UBE2D1_HuR17" in the figures), HA_UBE2D1_Linker2_HuR8 (labeled "UBE2D1_HuR8" in the figures), HA_HuR17_Linker2_UBE2D1 (labeled "HuR17_UBE2D1" in the figures), and HA_HuR8_Linker2_UBE2D1 (labeled "HuR8_UBE2D1" in the figures) fusion proteins (Figures 11 and 12). In certain instances, HuR levels were reduced by as much as 90% compared to control levels observed for cells expressing HA_UBE2D1_Linker2_Cas9 (labeled "UBE2D1_Cas9" in the figures) and HA_Cas9_Linker2_UBE2D1 (labeled "Cas9_UBE2D1" in the figures) (Figures 11B and 12D).

[0263] conclusion These data are HH We demonstrate that UBE2D1 fusion constructs containing single-domain antibody (nanobody) binding domains can successfully degrade target HuR (primarily nuclear targets). Quantification of target HuR degradation, shown in Figures 11B, 11D, 12B, and 12D, suggests that 65-90% of the HuR protein was degraded, implying that nuclear HuR was included in the degraded fraction.

[0264] Materials and Methods for Examples 1-7 Generation of lentiviral particles 5 x 10 HEK293FT cells 5 T25 flask, or 1 x 10 cells / flask 5Cells were seeded per well into 6-well plates in complete medium containing: Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% v / v heat-inactivated and gamma-irradiated fetal bovine serum (FBS; SAFC), 1% v / v sodium pyruvate (100x; Sigma), 1% v / v non-essential amino acids (100x; Invitrogen), 1% v / v Glutamax-1 (100x; Invitrogen), and Geneticin (G418) (final concentration 0.35 mg / mL; Invitrogen). Cells were incubated at 37°C and 5% CO2 for 3 days to allow attachment and 80% confluence. After this incubation period, the medium was removed and replaced with complete medium in the absence of Geneticin. For each lentiviral generation, the following was prepared:

[0265] [Table 2]

[0266] Different reagent volumes were used depending on the scale of lentiviral production. For further details, see the table above. One volume of dilution medium (OptiMEM; Invitrogen) was combined with pPACKH1 DNA (Cambridge Bioscience) and the gene of interest plasmid DNA (in the pCDH_puro lentiviral plasmid vector). For each transfection, a second volume of OptiMEM was mixed with Lipofectamine 2000 (Invitrogen) at room temperature for 5 minutes. The diluted plasmid mixture was then combined with the diluted Lipofectamine 2000 mixture and incubated at room temperature for 20 minutes. After this incubation period, HEK293FT cells were added and incubated at 37°C and 5% CO2 for 48 hours. After this incubation period, supernatants were collected from each cell sample and the presence of lentiviral particles was confirmed using Lenti-X™ GoStix™ Plus according to the manufacturer's instructions (Takara Bio). The supernatant containing the lentiviral particles was then filtered through a 0.22 μm pore filter into a sterile Steriflip (Millipore) tube before use.

[0267] Transduction of cells with lentivirus Ad293, MDA-MB-231, U20S, HCT116, HeLa, and HPAC cells were grown in 6-well plates in 2 mL of appropriate medium to achieve approximately 60-80% confluence. Prior to lentiviral transduction, all medium was removed and replaced with 2 mL of RPMI (Invitrogen) containing 10% FBS containing 16 μg / mL polybrene (final concentration: 8 μg / mL; Sigma-Aldrich). Two mL of supernatant containing lentiviral particles prepared as described above was added to each well. Cells were then incubated at 37°C and 5% CO2 for 24 h, after which the medium was replaced with fresh complete medium for each cell type. Cells were then incubated for an additional 24 h at 37°C and 5% CO2 before the addition of the selection antibiotic (puromycin; Thermo-Fisher Scientific). Puromycin was added at 2 μg / mL for Ad293, MDA-MB-231, U20S, and HPAC cells, 4 μg / mL for HCT116 cells, and 10 μg / mL for HeLa cells. Cells were maintained in the relevant medium containing antibiotics at 37°C, 5% CO2, until enough cells could be collected for Western blot analysis. Cell samples included pools of transduced cells.

[0268] Western blot analysis and quantification The medium was removed from the transduced cells and washed with PBS. Cells were harvested using Accutase (Sigma) and incubated at 37°C for 3 minutes. The Accutase was then neutralized by the addition of complete medium. The cell suspension was then collected and centrifuged at 1200 rpm (300 x g) for 5 minutes to pellet the cells. The cell pellet was washed with PBS and transferred to a 1.5 mL Eppendorf tube. These tubes were then centrifuged at 1200 rpm (300 x g) for 5 minutes, and the supernatant was discarded. The cell pellet was lysed in RIPA lysis buffer (Thermo Fisher Scientific) containing a 1:100 dilution of a protease and phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was incubated on ice for 30 minutes and then clarified by centrifugation at 10,000 rpm (17,000 x g) for 10 minutes at 4°C. The supernatant was collected in a new 1.5 mL Eppendorf tube and stored at -80°C. The protein concentration of each lysate was determined by BCA assay (Pierce / Thermo Fisher Scientific according to the manufacturer's instructions). 40 μg of lysate from each cell line was then loaded per well onto a 4-12% BOLT gel (Thermo Fisher Scientific) and run at 200 V for 25 minutes before being transferred to a membrane using iBlot according to the manufacturer's instructions (Thermo Fisher Scientific). The membrane was then blocked in Odyssey blocking buffer (Li-cor) and subjected to Western blot analysis using the appropriate antibodies (see Table 2 below).

[0269] [Table 3]

[0270] Blots were then visualized on an Odyssey system according to the manufacturer's instructions (Li-cor), and densitometry of Western blot bands was measured using Image Studio software according to the manufacturer's instructions (Li-cor).

[0271] Table 4

[0272] Table 5

[0273] Table 6

[0274] Table 7

[0275] Table 8

[0276] Table 9

[0277] Table 10

[0278] Table 11

[0279] Table 12

[0280] Table 13

[0281] Table 14

[0282]

Table 15

[0283] Table 16

[0284] Table 17

[0285] Table 18

[0286] Table 19

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[0288] Table 21

[0289] Table 22

[0290] Table 23

[0291] Table 24

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[0293] Table 26

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[0296] Table 29

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[0298] Table 31

[0299] Table 32

[0300] Table 33

[0301] Table 34

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[0303] Table 36

[0304] Table 37

[0305] Table 38

[0306] Table 39

[0307] Table 40

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[0314] Table 47

[0315] Table 48

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[0319] Table 52

[0320] Table 53

[0321] Table 54

[0322] Table 55

[0323] Table 56

[0324] Table 57

[0325] Table 58

[0326] Table 59

[0327] Table 60

[0328] Table 61

[0329] Table 62

[0330] Table 63

[0331] Table 64

[0332] Table 65

[0333] Table 66

[0334] Table 67

[0335] Table 68

[0336] Table 69

[0337] Table 70

[0338] Table 71

[0339] Table 72

[0340] Table 73

[0341] Table 74

[0342] Table 75

[0343] Table 76

[0344] Table 77

[0345] Table 78

[0346] Table 79

[0347] Table 80

[0348] Table 81

[0349] Table 82

[0350] Table 83

[0351] Table 84

[0352] Table 85

Claims

1. A molecule, (a) a regulatory domain comprising a human E2 enzyme comprising an E2 ubiquitin or ubiquitin-like conjugation domain; and (b) a targeting domain capable of targeting the regulatory domain to a substrate; and Including, A molecule wherein the human E2 enzyme is selected from the group consisting of UBE2D1 (UbcH5A) having the amino acid sequence of SEQ ID NO:4 and UBE2D2 (UbcH5B) having the amino acid sequence of SEQ ID NO:

5.

2. The molecule of claim 1 , wherein the targeting domain binds to the substrate.

3. 3. The molecule of claim 1 or 2, wherein the targeting domain is any one of a monobody, nanobody, antibody, antibody fragment, scFv, intrabody, minibody, scaffold protein, designed ankyrin repeat protein (DARPin), peptide binder, and ligand binding domain.

4. The molecule of any one of claims 1 to 3, wherein the targeting domain and / or the regulatory domain does not contain a lysine residue.

5. 5. The molecule of any one of claims 1 to 4, wherein the targeting domain has an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 126-135, 138-139, 257.

6. The molecule of any one of claims 1 to 5, wherein the substrate is an intracellular polypeptide.

7. 7. The molecule of any one of claims 1 to 6, wherein the substrate is localized in one or more of the plasma membrane, cytoplasm, nucleus, endosome, endoplasmic reticulum, mitochondria and Golgi apparatus.

8. The molecule of any one of claims 1 to 7, wherein the substrate is localized in the nucleus.

9. 8. The molecule of any one of claims 1 to 7, wherein the substrate is an oncogenic protein, a signaling protein, a GPCR, a post-translational modification protein, an adhesion protein, a receptor, a cell cycle protein, a checkpoint protein, a viral protein, a prion protein, a bacterial protein, a parasitic protein, a fungal protein, a DNA binding protein, a structural protein, an enzyme, an immunogen, an antigen, and / or a pathogenic protein.

10. 10. The molecule of any one of claims 1 to 9, wherein the substrate is selected from the group consisting of Ras, KRas, SHP2, human rhinovirus (HRV) protease 3C, muscarinic acetylcholine receptor 2 (M2R), beta-2 adrenergic receptor (β2-AR), cross-binding endonuclease MUS81 (MUS81), and human antigen R (HuR).

11. The molecule of any one of claims 1 to 10, wherein the regulatory domain and the targeting domain are linked by a linker.

12. The linker may be selected from the group consisting of peptides GGGGS (SEQ ID NO: 146), GGGGSGGGGSGGGGGS (SEQ ID NO: 145), LEGGGGGSSR (SEQ ID NO: 141), LEGGGGSGGGGGSGGGGSSR (SEQ ID NO: 142), AAAGGGGSGGGGGSGGGGSGT (SEQ ID NO: 143), GGGGG (SEQ ID NO: 144), LEGGSR (SEQ ID NO: 211), LEGGGSGGGSSR (SEQ ID NO: 212), LEGGGGSGGGGSSR (SEQ ID NO: 213), LEGGGSGGGSGGGSSR (SEQ ID NO: 214), LEGGGGSGGPSGGGGPSGSR (SEQ ID NO: 215), LESNGG 12. The molecule of claim 11, comprising GGSPAPAPGGGGGSGSSR (SEQ ID NO: 216), LEGGGGSYPYDVPDYASGGGGGSSR (SEQ ID NO: 217), TGGSAGGSGGSAGGSGGSAGGSGGSA (SEQ ID NO: 218), AGSGGSTGSGGSPTPSSTSGGSTGGAS (SEQ ID NO: 219), AGSGGSGGSGGSGNSSSTSGGSGGAS (SEQ ID NO: 220), GGSPVPSTPGGGSGGGGSGGSPVPSTPGS (SEQ ID NO: 221), or SPGTGSPGTGSPGTGSPGGTGSPG (SEQ ID NO: 222).

13. The molecule of any one of claims 1 to 12, which is a fusion polypeptide.

14. The molecule of any one of claims 1 to 13, wherein the regulatory domain is N-terminal to the targeting domain.

15. The molecule of any one of claims 1 to 13, wherein the regulatory domain is C-terminal to the targeting domain.

16. 16. The molecule of any one of claims 1 to 15, further comprising a detectable marker that does not contain a lysine residue, said detectable marker being a hemagglutinin tag or a Glu-Glu epitope tag.

17. 17. The molecule of any one of claims 1 to 16, wherein the molecule is a protein having the amino acid sequence of any one of SEQ ID NOs: 158-167, 171, 194, 195, 202-204, 236-248, 253, 254 and 267.

18. The molecule of any one of claims 1 to 17, wherein the molecule comprises a subcellular localization signal, a nuclear localization signal, a mitochondrial localization signal, or an endosomal localization signal.

19. 19. The molecule of any one of claims 1 to 18, wherein the molecule is capable of reducing the amount of the substrate by at least 20% compared to the amount of the substrate in the absence of the molecule, and wherein the molecule reduces the amount of the substrate in a cell by at least 20% compared to the amount of the substrate in an otherwise substantially identical cell that does not contain the molecule.

20. A compound comprising (i) a molecule according to any one of claims 1 to 19 and (ii) a targeting moiety capable of targeting said molecule to a cell.

21. 21. The compound of claim 20, wherein the targeting moiety is a binding partner or an antibody.

22. 22. The compound of claim 20 or 21, wherein the targeting moiety is a polypeptide fused to the molecule.

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