Activity-based probes

Abstract

The present invention relates to activity-based probes (ABPs) suitable for probing interactions between one or more biological molecules, as well as to their production, and use of them for profiling interactions between one or more biological molecules. The ABP may be a conjugate of ubiquitin (Ub) or a ubiquitin-like protein with an E2 conjugating enzyme (E2). The E2~Ub conjugate may be prepared with a suitably activated ubiquitin or ubiquitin-like protein and E2, and may be used to profile the activity of an E3 ligase (E3) from the ubiquitin or ubiquitin-like (Ubl) systems.

Description

[0001] ACTIVITY-BASED PROBES

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to activity-based probes (ABPs) suitable for probing interactions between one or more biological molecules, as well as to their production, and use of them for profiling interactions between one or more biological molecules. The ABP may be a conjugate of ubiquitin (Ub) or a ubiquitin-like protein with an E2 conjugating enzyme (E2). The E2~Ub conjugate may be prepared with a suitably activated ubiquitin or ubiquitin-like protein and E2, and may be used to profile the activity of an E3 ligase (E3) from the ubiquitin or ubiquitin-like (Ubl) systems.

[0004] BACKGROUND OF THE INVENTION:

[0005] Conjugation of the small protein ubiquitin (Ub) to protein substrates (known as ubiquitination) controls virtually all cellular processes. E3 ligases (E3s) operate at the end of an E1-E2-E3 enzyme cascade and conjugate ubiquitin to substrates. Small molecule modulation of E3 activity has great potential for disease treatment and E3 co-optation / recruitment is the premise of targeted protein degradation involving PROTAC (proteolysis-targeting chimera) molecules. Ubiquitin-like proteins (e.g. ISG15, SUMO and UFM1 ) are also conjugated to substrates via cognate yet analogous E1-E2- E3 machinery.

[0006] The ubiquitin / ubiquitin-like conjugation cascade is initiated by E1 enzymes that pass the Ub / Ubl carboxy terminus to an E2 enzyme to form a thioester-linked E2 (E2~Ub) conjugate. E2~Ub is then engaged by an E3, and ~50 ubiquitin E3s are known to contain an active site cysteine nucleophile. This facilitates an essential transthiolation (also known as thioesterification) reaction to form a thioester-linked E3 conjugate (E3~Ub).

[0007] E3 transthiolation is also regulated and dysregulated in disease and which E3s demonstrate transthiolation is poorly understood. Indeed, subtypes that have emerged in recent years have exposed new cellular paradigms and strategies for disease treatment. Despite the discovery of Ubls and their cognate E2s, the E3s for some remain unknown. Consequently, activity-based probes (ABPs) that measure E3 transthiolating activity have proven to be of great value. Additionally, the activities of multiple E3s, with different binding affinities, are able to be monitored in parallel, due to the covalent binding inherent to E3 function. This comprehensive profiling technique is a powerful way of surveying the E3 activity landscape across an array of experimentally or medically important conditions.

[0008] Extensive protein-protein interactions are involved when an E2~Ub conjugate recruits an E3 ligase. Once E3 is bound, the transthiolation step involves a nucleophilic addition of an E3 cysteine residue to an electrophilic E2~Ub thioester. Naturally, a probe to measure E3 transthiolating activity would comprise an E2~Ub conjugate with an electrophilic group. Such E2~Ub conjugates form the basis of specific E3 ABPs and have enabled:

[0009] (i) the dissection of E3 regulatory mechanisms; (ii) insights into disease aetiology; (iii) E3 discovery; and (iv) the stabilisation of their enzymatic intermediates for structural analysis.

[0010] The synthesis of E2~Ub ABPs containing an electrophile, however, presents a distinct protein synthetic challenge. Although semisynthetic procedures for E2~Ub ABPs have been developed, they incorporate highly reactive dehydroalanine (Angewandte Chemie International Edition, vol. 60, page 17171 , 2021 ) or vinyl ketone (Nature, vol. 590, page 671 , 2021 ) electrophiles. In such E2~Ub ABPs, the electrophilic centres are in a compromised position, which can result in off-target labelling.

[0011] Furthermore, it is known in the art that more modestly-electrophilic E2~Ub ABPs comprising a vinyl sulfide electrophile can be prepared via tosyl-substituted doubly- activated enes (TDAEs) (EP 3201352). However, the preparation of such ABPs requires a convergent multistep synthetic sequence that necessitates the incorporation of a triazole moiety, introduced to mediate the convergence. Not only is the synthesis expensive and complex, but the triazole moiety likely results in slow labelling kinetics (Nature Chemical Biology, vol. 12, page 324, 2016). That is to say, the extensive proteinprotein interactions inherent to native E3 transthiolating activity are likely obscured in the aforementioned ABPs by the triazole “scar” left during synthesis. This moiety likely impedes the ability of E3s to react with such ABPs.

[0012] In summary, current generation ABPs are limited by:

[0013] (i) off-target labelling;

[0014] (ii) multistep synthetic procedures required to produce them; and

[0015] (iii) slow E3 labelling kinetics. There is a need in the art to provide alternative ABPs, preferably alternatives that are not limited by one or more of the limitations described above. The present invention addresses that need.

[0016] SUMMARY OF THE INVENTION:

[0017] The present inventors have prepared novel activity-based probes (ABPs) for use in profiling interactions between biological molecules. The inventors have found that their novel ABPs are, surprisingly, highly target specific, synthetically tractable, and possess fast labelling kinetics.

[0018] Viewed from a first aspect, therefore, there is provided a compound corresponding to formula (I): wherein X and Y are each independently a biological molecule;

[0019] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

[0020] The compound of formula (I) may be prepared by reacting a biological molecule comprising a thiol moiety (e.g. protonated S-Y, i.e. HS-Y) with a precursor compound comprising a leaving group in place of S-Y.

[0021] Thus, viewed from a second aspect, there is provided a compound corresponding to formula (II): wherein X is a biological molecule;

[0022] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile or halo; and LG is a leaving group.

[0023] Viewed from a third aspect, there is provided a method for preparing a compound as defined in claim 1 , the method comprising contacting a biological molecule comprising a thiol moiety (e.g. protonated S-Y, i.e. HS-Y) with a compound of formula (II): wherein X is a biological molecule;

[0024] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and LG is a leaving group.

[0025] As described above, the inventors have found that their novel ABPs, when used to profile interactions between biological molecules, are surprisingly highly target specific, synthetically tractable, and possess fast labelling kinetics.

[0026] Thus, viewed from a fourth aspect, there is provided a method of detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, the method comprising providing a first compound of formula (I): wherein X and Y are each independently a biological molecule; Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and contacting the compound of formula (I) with one or more biological molecules, such that any of the one or more biological molecules with transthiolation activity are able to react with the compound of formula (I) to form a conjugate; and detecting the formation, or lack thereof, of conjugates.

[0027] Viewed from a fifth aspect, there is provided use of a compound corresponding to formula (I) for detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, wherein formula (I) is: wherein X and Y are each independently a biological molecule;

[0028] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

[0029] DEFINITIONS

[0030] In the discussion that follows, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds according to the invention, is in general based on the rules of the IUPAC organisation for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IUPAC organisation is in conflict with a definition provided herein, the definition herein is to prevail. Furthermore, if a compound structure is in conflict with the name provided for the structure, the structure is to prevail. The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0031] The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0032] The term “alkyl” is well known in the art and defines univalent groups derived from alkanes by removal of a hydrogen atom from any carbon atom, wherein the term “alkane” is intended to define acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, wherein n is an integer >1. Alkyl groups may be C1-6alkyl groups. In some cases, alkyl groups are C1-4alkyl groups. C1-4alkyl refers to any selected from the group consisting of methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so-butyl and tert-butyl.

[0033] The term “haloalkyl” is also well known and defines univalent groups derived from alkyl groups by replacement of one or more hydrogen atoms from one or more carbon atoms with a halo group. Haloalkyl groups may comprise one or more different types of halo. For example, one or more independently selected from fluoro, chloro, bromo and iodo. In some cases, the haloalkyl is a fluoroalkyl.

[0034] Halo refers to a halogen radical. Typically, halo refers to any selected from fluoro, bromo, chloro and iodo. In some cases, halo refers to fluoro.

[0035] The term “nitrile” is well known in the art and refers to the -CN group, otherwise represented as -CEN.

[0036] The term “alkoxy” defines univalent groups derived from alcohols by removal of a hydrogen atom from an OH group, wherein the term “alcohol” is intended to define groups derived from alkanes by the replacement of a hydrogen atom with a hydroxy group. Often, alkoxy groups are C1-6alkoxy groups or C1-4alkoxy groups. The term “alkenyl” defines univalent groups derived from alkenes by removal of a hydrogen atom from any carbon atom, wherein the term “alkene” is intended to define acyclic branched or unbranched hydrocarbons having one carbon-carbon double bond and the general formula CnH2n, where n is an integer >2. C2-C4alkenyl refers to any one selected from the group consisting of ethenyl, prop-1 -enyl, prop-2-enyl, 1-methyl- ethenyl, but-1-enyl, but-2-enyl, but-3-enyl, 1-methyl-prop-1-enyl, 1-methyl-prop-2-enyl, 2-methyl-prop-1-enyl, and 2-methyl-prop-2-enyl.

[0037] The term “haloalkenyl” defines univalent groups derived from alkenyl groups by replacement of one or more hydrogen atoms from one or more carbon atoms with a halo group. Haloalkenyl groups may comprise one or more different types of halo. For example, one or more independently selected from fluoro, chloro, bromo, and iodo. In some cases, the haloalkenyl is a fluoroalkenyl.

[0038] The term “biological molecule” is well known in the art and relates to any molecule present in organisms that is involved in one or more typically biological processes. Biological molecules may include but are not limited to proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones. In some embodiments, the biological molecules (e.g. that of X and / or Y) comprises one or more selected from amino acids, peptides and proteins. Biological molecules may be extracted from their natural source. They may be produced by synthetic or biotechnological means. Biological molecules may be of an unnatural origin, have no known biological purpose, or may not be known to be involved in any biological process. They may be engineered or produced in such a way to differ from their natural counterparts, for example the biological molecule may be a variant or modified form of a natural biological molecule. This includes biological molecules derived from natural proteins that have been modified in some way, e.g. to comprise one or more mutations, additions, deletions, inversions etc.

[0039] The term “ubiquitin-like protein”, or Ub / Ubl, refers to any small protein that can be conjugated to a substrate protein through the modes of action of E1 , E2, and E3 enzymes, typically to tag the substrate protein for degradation. The term “ubiquitin” or “Ub” refers to a regulatory protein found in most tissues of eukaryotic organisms, of a size of about 8.6 kDa. Typically, ubiquitin refers to human ubiquitin, which has a structure that is well known in the art. For the avoidance of any doubt, any reference herein to the term “ubiquitin” or “Ub” does not exclude any reference or incorporation of the terms “ubiquitin-like protein” or “Ubl”. The same applies vice versa.

[0040] The term “E1 activating enzyme”, “ubiquitin-activating enzyme”, or “E1” refers to any protein that binds ubiquitin and transfers the ubiquitin to an E2 enzyme.

[0041] The term “E2 conjugating enzyme”, or “ubiquitin-conjugating enzyme”, or “E2” refers to any protein that obtains ubiquitin from an E1~Ub conjugate, and / or collaborates with an E3 ligase to transfer ubiquitin to a protein substrate.

[0042] The term “E3 ubiquitin ligase”, or “E3 ligase”, or “E3” refers to any protein that recruits an E2 conjugating enzyme that has been loaded with ubiquitin or a ubiquitin-like protein, recognizes a protein substrate, and assists or directly catalyses the transfer of ubiquitin, or the ubiquitin-like protein, from the E2 protein to the protein substrate.

[0043] The term “contacting” is used herein to refer to any one or more of the acts of combining, reacting, mixing, stirring, slurrying, blending, dissolving, treating, incubating, joining, supplying, passing over, flowing over, or otherwise contacted in some manner, in any order, and for any length of time.

[0044] The term “sulfonyl” is well known in the art and refers herein to compounds derived from sulfonic acids by the removal of a hydroxy group. The sulfonyl has a general formula of -SO2R, where R may be an organyl group such as an optionally substituted phenyl, optionally substituted naphthalenyl or an optionally substituted C1-6alkyl group (such as an optionally substituted methyl or ethyl). The optional substituents of the phenyl or napthalenyl may be any one or more selected from C1-6alkyl (such as methyl), C1-6haloalkyl (such as trifluoromethyl), halo (such as bromo or fluoro), nitro and diCi. ealkylamino (such as dimethylamino). The optional substituents of the C1-6alkyl may be any one or more selected from halo (such as fluoro), nitro and diC-i-ealkylamino (such as dimethylamino).

[0045] The term “leaving group” is well known in the art and defines an atom or group of atoms (charged or uncharged) that may become detached from the residual or main part of a compound as part of a reaction. The term includes atoms or groups of atoms that depart with or without a pair of electrons in a heterolytic bond cleavage. Atoms or groups that may act as leaving groups in reactions include but are not limited to halo, sulfonyl, sulfonate, sulfinyl, sulfinate, dinitrogen, dialkyl ether, water, nitrate, phosphate, thioether, amine, and ammonia. Further examples of leaving groups are 4-methylphenyl-1 -sulfonyl, 4-bromophenyl-1 -sulfonyl, 4-nitrophenyl-1 -sulfonyl, methanesulfonyl, trifluoromethanesulfonyl, 2 ,2 ,2-trif I u oroethy I- 1 -sulfonyl, 5-(dimethylamino)naphthalene- 1 -sulfonyl, iodo, bromo, and chloro.

[0046] In some cases, 4-methylphenyl-1 -sulfonyl may be referred to as “tosyl”, 4-bromophenyl- 1 -sulfonyl as “brosyl”, 4-nitrophenyl-1 -sulfonyl as “nosyl”, methanesulfonyl as “mesyl”, trifluoromethanesulfonyl as “triflyl”, 2, 2, 2-trifluoroethyl-1 -sulfonyl as “tresyl”, and 5- (dimethylamino)naphthalene-l -sulfonyl as “dansyl”.

[0047] The term “enantiomer” defines one of a pair of molecular entities that are mirror images of each other and non-superimposable, i.e., cannot be brought into coincidence by translation and rigid rotation transformations. Enantiomers are chiral molecules, i.e., are distinguishable from their mirror image.

[0048] The term “racemic” is used herein to pertain to a racemate. A racemate defines a substantially equimolar mixture of a pair of enantiomers, which typically comprises a pair of enantiomers in a ratio of about 1 :1.

[0049] The term “diastereoisomers” (also known as diastereomers) defines stereoisomers that are not related as mirror images.

[0050] The term “solvate” is used herein to refer to a complex comprising a solute, such as a compound or salt of the compound, and a solvent. If the solvent is water, the solvate may be termed a hydrate, for example a mono-hydrate, di-hydrate, tri-hydrate etc., depending on the number of water molecules present per molecule of substrate.

[0051] The term “isotope” is used herein to define a variant of a particular chemical element, in which the nucleus necessarily has the same atomic number but has a different mass number owing to it possessing a different number of neutrons.

[0052] As described above, in the first aspect there is provided a compound corresponding to formula (I): wherein X and Y are each independently a biological molecule; Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

[0053] In some embodiments, Y is an E2 conjugating enzyme (E2). Preferably, the E2 enzyme is selected from UBE2D1 , UBE2D2, UBE2D3 or UBE2L3.

[0054] In some embodiments, Z is -N(R1)-. In some embodiments, X is ubiquitin, and / or R1is H, and / or R2is H.

[0055] In some embodiments, the sulphur atom of S-Y (connected to the p-position - relative to the carbonyl - of a compound of formula (I)) is derived from a cysteine residue comprised within the biological molecule. The cysteine residue may be naturally occurring or the cysteine residue may be introduced to the biological molecule.

[0056] X and Y may be independent biological molecules connected only by the acrylohydrazide sulfide moiety depicted in formula (I). Alternatively, X and Y may relate to two independent moieties of one biological molecule, so as the acrylohydrazide sulfide moiety is bridging two parts of a biological molecule together. Where X and Y are independent biological molecules, if X and Y were to be connected to the same atom or group of a molecule, or connected directly together, they may be considered to form one whole biological molecule or a conjugate of two biological molecules. That is to say, the acrylohydrazide sulfide moiety may be considered to be an insertion to the structure of either a biological molecule or a conjugate of two biological molecules.

[0057] The inventors have identified compounds that are suitable for the preparation of the ABPs of the first aspect. As described above, the compound of formula (I) may be prepared by reacting a biological molecule comprising a thiol moiety (e.g. protonated S- Y, i.e. HS-Y) with a precursor compound comprising a leaving group in place of S-Y.

[0058] In the second aspect, there is provided a compound corresponding to formula (II): wherein X is a biological molecule;

[0059] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile or halo; and LG is a leaving group.

[0060] For the avoidance of doubt, any of the embodiments described in relation to moieties relevant to the second aspect, such as in relation to Z, R1, R2and X of the first aspect, may apply mutatis mutandis to the second aspect. For example, Z may be -N(R1)-, and / or X may be ubiquitin, and / or R1may be H, and / or R2may be H.

[0061] In some embodiments, LG is selected from the group consisting of chloro, bromo, or iodo.

[0062] In some embodiments, LG corresponds to formula (III): wherein R3is selected from the group consisting of C1-6alkyl; C1-6haloalkyl; phenyl; and naphthyl, optionally substituted one or more times with C1-6alkyl, C1-6alkoxy, halo, amino, alkylamino, or nitro. Preferably, R3is selected from the group consisting of methyl, trifluoromethyl, 4-methylphenyl, 4-nitrophenyl, 4-bromophenyl, 2,2,2-trifluoroethyl, and 5-(dimethylamino)naphthalyl. Typically, R3is 4-methylphenyl, i.e. LG is tosyl.

[0063] The present inventors have devised a traceless and unexpectedly efficient method to prepare ABPs. The third aspect provides a method for preparing a compound of formula (I), the method comprising contacting a biological molecule comprising a thiol moiety (e.g. HS-Y) with a compound of formula (II): wherein X is a biological molecule;

[0064] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and LG is a leaving group.

[0065] For the avoidance of doubt, any of the embodiments described in relation to moieties relevant to the third aspect, such as in relation to Z, R1, R2, X and Y of the first aspect and LD of the second aspect, may apply mutatis mutandis to the third aspect. For example, Z may be -N(R1)-, and / or X may be ubiquitin or ubiquitin-like protein, and / or R1may be H, and / or R2may be H, and / or Y may be E2, and / or LG may be of formula (III).

[0066] In some embodiments, the contacting comprises addition of the compound of formula (II) to the biological molecule comprising a thiol moiety. In alternative embodiments, the biological molecule comprising a thiol moiety is added to the compound of formula (II). The compound of formula (II) and the biological molecule comprising a thiol moiety may each independently be in a solution, suspension or slurry on contacting. In some embodiments, each are in solution, for example a buffer solution, on contacting. Alternatively, one may be in solution, such as a buffer solution, and the other may be added neat or in a concentrated sample. The buffer solution may comprise any one or more selected from Na2HPO4, HEPES, NaCI, and TCEP.

[0067] In some embodiments, the thiol moiety (SH) is within a cysteine residue. The cysteine residue may be naturally occurring or the cysteine residue may be introduced to the biological molecule.

[0068] It is an object of the present invention to provide ABPs to profile interactions between biological molecules. The fourth aspect provides a method of detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, the method comprising providing a compound of formula (I): wherein X and Y are each independently a biological molecule;

[0069] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and contacting the compound of formula (I) with one or more biological molecules, such that any of the one or more biological molecules with transthiolation activity, are able to react with the compound of formula (I) to form a conjugate; and detecting the formation, or lack thereof, of conjugates.

[0070] For the avoidance of doubt, any of the embodiments described in relation to moieties relevant to the fourth aspect, such as in relation to Z, R1, R2, X and Y of the first aspect, may apply mutatis mutandis to the fourth aspect. For example, Z may be -N(R1)-, and / or X may be ubiquitin or ubiquitin-like protein, and / or R1may be H, and / or R2may be H, and / or Y may be E2.

[0071] An interaction between a compound of formula (I) and one or more biological molecules may include but is not limited to a biochemical reaction. The biochemical reaction may be an addition or substitution reaction, that is to say, a compound of formula (I) and one or more biological molecules may join to form one or more new compounds, e.g., one or more conjugates. In some embodiments, the interaction is an addition reaction whereby a thiol moiety of a biological molecule undergoes addition to the p-position - relative to the carbonyl - of a compound of formula (I), resulting in a conjugate, with concurrent saturation of the a-p carbon-carbon double bond and the formation of a dithioacetal moiety. Such an addition reaction may be referred to as “transthiolation” or “thioesterification”.

[0072] Transthiolation may be understood to relate to any reaction, including biochemical reactions, whereby a thiol moiety of a first compound or biological molecule is transferred to a second compound or biological molecule. Such a transthiolation reaction may involve the transfer of a part or the entirety of the first compound or biological molecule to the second. That is to say, a conjugate may be formed. Transthiolation activity is thereby understood to relate to the propensity of a compound or biological molecule to undergo a transthiolation reaction. For example, a compound or biological molecule that has fast transthiolation kinetics thereby has high transthiolation activity. On the other hand, a compound or biological molecule that has no or very low transthiolation activity will either not undergo transthiolation or have transthiolation kinetics prohibitively slow to the extent that the transthiolation activity is undetectable.

[0073] In some embodiments, the contacting comprises adding a compound of formula (I) to the biological molecule. Alternatively, the biological molecule may be added to the compound of formula (I). The compound of formula (I) and the biological molecule may each independently be in a solution, suspension or slurry on contacting. In some embodiments, each are in solution, for example a buffer solution, on contacting. Alternatively, one may be in solution, such as a buffer solution, and the other may be added neat or in a concentrated sample. The buffer solution may comprise any one or more selected from Na2HPO4, HEPES, NaCI, and TCEP.

[0074] The detecting of interactions may be carried out using methods such as gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), fluorescence based methods (e.g. fluorescence polarization, or fluorescence resonance energy transfer), magnetic separation, nuclear magnetic resonance (NMR) spectroscopy, immunological separation, gel filtration chromatography, affinity chromatography, column chromatography, displacement chromatography, electro-chromatography, gas chromatography (GC), high-performance liquid chromatography (HPLC), liquidchromatography mass-spectrometry (LCMS), ion chromatography, micellar electrokinetic chromatography, thin-layer chromatography (TLC), centrifugation, adhesion, flow cytometry, or other techniques known to the skilled person. Typically, the gel electrophoresis is SDS polyacrylamide gel electrophoresis.

[0075] SDS polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used method for protein analysis. It can separate proteins into different bands according to molecular weight in an electric field under denaturing conditions. SDS-PAGE can enable the separation and quantification of one or more biological molecules contained within a mixture.

[0076] In some embodiments, the detecting of interactions may comprise the detection of the formation of a compound or biological molecule. Additionally, the aforementioned methods of detection may detect the lack of formation of a compound or biological molecule. That is to say, they may detect the lack of a signal and thus a lack of interaction.

[0077] In some embodiments, the interaction between biological molecules to be detected is between Ub / Ubl-activating enzymes (E1s) and E2 conjugating enzymes (E2s); or between E2s and E3 ligases (E3s).

[0078] The skilled person will appreciate that many different E1 , E2, E3, and Ub / Ubl proteins and isozymes are known and may be used in the present invention.

[0079] The fifth aspect provides for the use of a compound corresponding to formula (I) for detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, wherein formula (I) is: wherein X and Y are each independently a biological molecule;

[0080] Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

[0081] For the avoidance of doubt, any of the embodiments described in relation to moieties relevant to the fifth aspect, such as in relation to Z, R1, R2, X and Y of the first aspect and the contacting and detecting of the fourth aspect, may apply mutatis mutandis to the fifth aspect. For example, Z may be -N(R1)-, and / or X may be ubiquitin, and / or R1may be H, and / or R2may be H, and / or Y may be E2, and / or the compound of formula (I) and the one or more biological molecules may each be in solution, for example a buffer solution, and / or the detecting of interactions may be by SDS-PAGE.

[0082] The compounds of the present invention may be modified by incorporation of a tag.

[0083] The term “tag” as used herein denotes a biochemical marker or label, that is to say an easily recognisable chemical moiety, for example a protein, peptide, or small molecule, that is covalently attached to the compound. The tag may be attached to or form part of the biological molecules X and / or Y of the compounds. Numerous tags are known to the skilled person, and include but are not limited to affinity labels, epitope tags, fluorophores, biotin, biotinyl-derivatives and surrogates, photocrosslinking groups, peptide tags and radioactive labels.

[0084] The compounds of the invention may exist in different stereoisomeric forms. All stereoisomeric forms and mixtures thereof, including enantiomers and racemic mixtures, are included within the scope of the invention. Such stereoisomeric forms include enantiomers and diastereoisomers. Individual stereoisomers of compounds of the invention, i.e., associated with, for example mixed with, less than 5%, preferably less than 2% and in particular less than 1 % of the other stereoisomer, are included. Mixtures of stereoisomers in any proportion, for example a racemic mixture comprising substantially equal amounts of two enantiomers are also included within the invention.

[0085] Additionally, the compounds of the present invention are depicted with a particular arrangement of the atoms or groups about the a-p (relative to the carbonyl) carbon- carbon double bond. That is to say, just one stereochemical configuration is depicted in formula (I) and formula (II). This does not exclude any alternative stereochemical configurations of the compounds from the invention. For example, in some embodiments of the present invention, the stereochemical configuration may be assigned as ‘trans’ or ‘E’. In other embodiments, the stereochemical configuration may be assigned as ‘cis’ or ‘Z’. The invention may comprise a mixture of any proportion of trans and cis forms of the same compound.

[0086] Also included are solvates and isotopically-labelled compounds of the invention. Isotopically-labelled compounds are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as2H (i.e., deuterium),3H,13C,14C,15N,18O,170,35S,18F, and36CI, respectively.

[0087] A protium atom (H) is a hydrogen atom with zero neutrons. A deuterium atom (D or2H) is a hydrogen atom with one neutron. Naturally occurring hydrogen contains about 0.02 molar per cent deuterium and 99.98% protium. Physical chemical properties between protium and deuterium are small but measurable. Deuterium is slightly less lipophilic than protium, has a smaller molar volume and carbon-deuterium bonds are shorter than carbon-protium bonds. Deuterium keeps the 3D surface, shape and steric flexibility of a molecule unaltered compared to H. A ratio of deuteriurmprotium in a compound greater than 1 :99 is considered to be greater than that found naturally in hydrogen. In particular embodiments, where a moiety is specified as being “H”, the ratio of deuterium:protium at this position is greater than the natural isotopic abundance of deuterium, i.e. the percentage of deuterium found at his position of the compounds of the invention is greater than its natural isotopic abundance in hydrogen, which is about 0.02 mol%.

[0088] All amorphous and crystalline forms of the compounds of the invention are included.

[0089] BRIEF DESCRIPTION OF THE FIGURES:

[0090] The invention may be further understood by reference to the non-limiting figures. Figure 1 shows: A) Proposed reaction of hydrazide-linked ubiquitin-TDAE conjugate with a recombinantly produced E2 enzyme to furnish an E2~Ub ABP; and B). Use of the small molecule A / -acetyl-g lycine hydrazide as a proxy to establish whether anhydride acylation of a ubiquitin hydrazide would produce the desired Ub-TDAE conjugate.

[0091] Figure 2 shows: A) The native E2~Ub conjugate; and B) first and second generation vinyl sulfide E2~Ub ABPs.

[0092] Figure 3 shows: Assessment of activity-based profiling of the HECT E3 NEDD4L and its inactive counterpart containing a C922A mutation. The UBE2D3~Ub-1-75ABP (12 pM) was incubated with E3 (3 pM) for the indicated time point and temperature.

[0093] Figure 4 shows: A) Coomassie stained SDS-PAGE gel depicting the conjugation of species of formula (II), where X is the protein SUMO1 missing the C-terminal residue present in the processed form. The EWG in this case is a tosyl group. Y is the E2 conjugating enzyme Ubc9. Mixing in biological buffer results in the formation of probe molecule E2-SUMO1 , which is of formula (I), were X is SUMO1 and Y is Ubc9. Some of the non-catalytic cysteines in Ubc9 were mutated to ensure specific conjugation of X to the active site cysteine. The protein conjugate (I) was purified by size exclusion chromatography. B) The same preparation was carried out for the SUMO2 paralogue. In both cases, the SUMO proteins carry a biotin moiety conjugated to an introduced N- terminal cysteine residue. C) SDS-PAGE gel confirming the SUMO E1 activating enzyme subunit containing the catalytic cysteine (SAE2) has been labelled with SUMO ABP.

[0094] Figure 5 shows: A) Coomassie stained SDS-PAGE gel depicting the conjugation of species of formula (II) where X is the protein ISG15 lacking the N-terminal Ubl fold (ISG15CTD). The EWG in this case is a tosyl group. Y is the E2 conjugating enzyme UbcH8. Mixing in biological buffer results in the formation of probe molecule E2~ ISG15CTD, which is of formula (I), were X is ISG15CTDand Y is UbcH8. The non- catalytic cysteines in UbcH8 were mutated to ensure specific conjugation of X to the active site cysteine. B) The ABP conjugate was purified from the crude reaction by size exclusion chromatography. C) Coomassie stained SDS-PAGE gel confirming the E2~ISG15CTDABPS underwent activity-dependent covalent labelling of the catalytic HECT domain from the ISG15 E3 HERC5. DETAILED DESCRIPTION OF THE INVENTION:

[0095] The inventors sought a chemical moiety and a method to construct such a moiety that could form an ABP with:

[0096] (i) limited off-target labelling;

[0097] (ii) synthetic tractability; and

[0098] (iii) fast E3 labelling kinetics.

[0099] The inventors found that a thioacrylohydrazide moiety unexpectedly fit the requirements. Accordingly, the inventors devised a clever synthetic sequence of surprising brevity. This started from a C-terminal protein hydrazide, which could be obtained from a Ub-intein fusion. The inventors postulated that an appropriately designed anhydride - 2,3- dibromopropanoic anhydride (1 ) - would then enable quick access to a Ub-TDAE (tosyl doubly activated ene) conjugate. Such a conjugate could then react with an E2 enzyme which could furnish an E2~Ub ABP with a thioacrylohydrazide electrophilic group.

[0100] It is known in the art that the native E2~Ub / Ubl conjugate consists of Ub / Ubl thioester- bonded via its C-terminus to the active site cysteine in an E2 enzyme (Figure 2A). Furthermore, HECT and HECT-like E3s contain an active site cysteine that attack the thioester bond in E2~Ub forming a tetrahedral intermediate, before the transfer of Ub to the E3 active site. The inventors consequently reasoned that the proposed strategy could furnish an E2~Ub / Ubl ABP with improved isostery to said native E2~Ub / Ubl conjugates (Figure 1A).

[0101] To establish feasibility of the proposed strategy, model reactions were carried out on A / -acetyl-glycine hydrazide. The reaction scheme began with acylation of the model hydrazide with 2,3-dibromopropanoic anhydride 1 to form the dibromo intermediate 2 (Figure 1 B). Anhydride 1 was synthesized by the treatment of 2,3-dibromopropanoic acid with 0.5 equivalents of dicyclohexocarbodiimide (DCC) and the crude product was isolated. In pH 3 phosphate-buffered DMF, the expected dibromo adduct 2 was obtained after the reaction of A / -acetyl-glycine-hydrazide with 1 for 90 minutes at 23 °C, which was purified by HPLC. It was then investigated whether 2 could be converted in situ to the desired TDAE by substitution of the terminal bromide and concerted elimination of HBr when reacted with sodium p-toluenesulfinate (TsNa). Dibromo adduct 2 was reacted with 50 equivalents of TsNa in pH 7.5 phosphate buffer. LCMS and NMR confirmed the formation of the trans TDAE conjugate 3. Conjugate 3 was incubated with A / -acetyl-cysteine, which rapidly reacted in aqueous pH 7.5 phosphate buffer to form thioacrylohydrazide conjugate 4. This result confirmed that the proposed strategy was indeed feasible; that is to say, a Michael type-addition-elimination with a cysteine residue to furnish a conjugate containing a thioacrylohydrazide electrophile was successful, thereby generating a new class of ABP.

[0102] With the novel strategy for TDAE conjugate formation validated, it was then applied to the production of E2~Ub ABPs. To accommodate the introduced thioacrylohydrazide moiety, whilst ensuring optimal isostery with a native E2~Ub, two different C-terminal Ub hydrazides were prepared, with C-terminal Gly76, or, Gly75 and Gly76 deleted (Ub-i-75- NHNH2 and Ubi-74-NHNH2, respectively). These would produce an E2~Ub where relative to the native conjugate, the number of bond lengths was increased by two or reduced by one, respectively (Figure 2B).

[0103] Initially, C-terminal ubiquitin thioesters were produced from the corresponding Ub-intein fusions which were expressed in E. coli, purified against chitin resin, and then incubated in aqueous pH 5 sodium acetate buffer containing 100 mM 2-mercaptoethanesulfonic acid (MESNA). In situ conversion of the thioesters to the hydrazides was carried out by adjusting the supernatants to pH 4 with HCI and then stirring with 100 mM hydrazine monohydrate for 1 h at 23 °C. Ubi-75-NHNH2 and Ubi-74-NHNH2 were then purified by ion exchange chromatography and positive fractions identified by LCMS. The pH was further reduced to pH 3, followed by the addition of 200 equivalents of 1 . The reaction was stirred for 1 h at 23 °C which resulted in the quantitative depletion of the hydrazide and formation of the anticipated dibromo adduct. However, also observed was the unexpected formation of a species with a mass corresponding to an HBr elimination product.

[0104] The inventors surmised elimination was disfavoured in the model reactions because the presence of DMF reduced the proticity of the solvent. It was reasoned that the eliminated product might also react with TsNa to form the desired TDAE conjugate. To test this, 2 was induced to elimination of HBr by treatment with triethylamine, which generated a major product being the 2-bromo geminal vinyl (J coupling constant = 2.9 Hz) species and a minor species corresponding to the 2-bromo cis vinyl species. The crude product mix was reacted with TsNa and a single product was formed that was purified by RP- HPLC. NMR analysis revealed the formation of 3, indicating that reaction of dibromo conjugate 2 - or its mixed elimination products - with TsNa converges to the desired trans TDAE conjugate.

[0105] With this uncertainty resolved, the two products derived from Ub-1-75-NHNH2 and Ubi-74-NHNH2 were collectively purified by HPLC, lyophilized, re-suspended in denaturing buffer (50 mM Na2HPO4pH 7.5, 6 M guanidinium chloride) and vigorously stirred with 2000 equivalents of TsNa. LCMS confirmed the exclusive production of the Ub-TDAE products (Ubws-TDAE and Ub-i-74-TDAE), which were purified by a second round of HPLC and lyophilized. To assemble the E2~Ub ABPs, the Ubws-TDAE and Ubi -74-TDAE products were dissolved in DMSO, then refolded by dilution into phosphate- buffered saline. Folded Ub-TDAE conjugates were next incubated with an excess of either the E2 UBE2D3 or UBE2L3, both mutants containing a single cysteine at the active site position, for 2 h at 30 °C in aqueous phosphate buffered saline.

[0106] Loading of the Ub-TDAE variants onto the E2s went to near completion and the E2~Ub ABPs were purified by size exclusion chromatography and characterized by LCMS. By reacting the ubiquitin-TDAE with two different E2 enzymes, the production of two complementary E3 ABPs has been demonstrated by the inventors. Through the novel exploitation of C-terminal hydrazide acylation, the inventors have prepared the requisite ubiquitin-TDAE conjugate with minimal perturbation of the native ubiquitin structure.

[0107] The inventors then tested the specificity and efficiency of this new class of E2~Ub ABP with the purified catalytic HECT domain from NEDD4L. Strikingly, ABP labelling was observed at the first time point which was the ~1 minute it took to prepare reactions before quenching, even at 0 °C (Figure 3). No labelling of an active site cysteine mutant of the NEDD4L catalytic domain was observed, despite containing 4 non-catalytic cysteines. Specificity towards the E3 active cysteine was maintained even after prolonged incubation and elevated temperature.

[0108] EXPERIMENTAL:

[0109] General experimental information: Laboratory chemicals, solvents, and small molecule reagents for organic synthesis were obtained from Sigma Aldrich and were used as received.

[0110] LCMS samples were analysed on Agilent 1200 system coupled to a 6100 series quadrupole mass spectrometer, with a C3 reversed-phase column running a gradient of 10% buffer A (H2O + 0.05% TFA) to 70% buffer B (acetonitrile + 0.05% TFA) over 20 minutes. Size exclusion and ion exchange chromatography were carried out with an Akta Pure FPLC system (Cytiva). Proteins were expressed in E coli strain BL21 (DE3) (Merck).

[0111] Synthesis of 2,3-dibromopropanoic anhydride (1 ):

[0112] 10 g of 2,3-dibromopropionic acid was dissolved in 50 mL DCM in a 100 mL RB flask under an argon atmosphere and the flask was cooled to 0 °C with an ice bath. DCC (0.5 equiv., 4.45 g, 21 .56 mmol) was added in portions at 0 °C. The clear solution turned milky within minutes due to the formation of DCU. Stirring was continued overnight, during which the reaction was allowed to reach room temperature. The mixture was filtered on a Celite pad to remove DCU and washed with DCM. The organic phase was then evaporated under reduced pressure. To remove traces of DCU, the residue was re- dissolved in MeCN and filtered a second time on Celite, and evaporated under reduced pressure to afford anhydride 1 as an orange oil. 1 H NMR (400 MHz, Chloroform-d) δ 4.58 (ddd, J = 11.0, 4.5, 0.6 Hz, 2H), 3.95 (ddd, J = 11.3, 10.0, 0.9 Hz, 2H), 3.75 (ddd, J = 10.4, 4.5, 0.6 Hz, 2H).

[0113] Synthesis of N-(2-(2-(2,3-dibromopropanoyl)hvdrazineyl)-2-oxoethyl)-2~ Dibromo anhydride 1 (300 mg, 0.69 mmol) was dissolved in 2 mL DMF. Then, 400 mg A / -acetyl-glycine hydrazide (5 equiv., 452 mg, 3.45 mmol) was dissolved in 4 mL of buffer (100 mM Na2HPO4pH 3, 150 mM NaCI). Then, at room temperature, the anhydride was added dropwise under vigorous stirring to the N -acetyl-glycine hydrazide solution, during which a precipitate formed. The reaction was allowed to stir at RT for 90 min, after which the solids were removed by centrifugation, and the crude product was purified by RP- HPLC (C18 column, gradient 5-50% MeCN / H2O over 20 min. NMR indicated complete (E) olefin.

[0114] 1 H NMR (400 MHz, DMSO-d6) δ 10.59 (d, J = 2.4 Hz, 1 H), 10.27 (d, J = 2.4 Hz, 1 H), 8.11 (t, J = 5.9 Hz, 1 H), 4.67 (dd, J = 9.5, 6.1 Hz, 1 H), 3.96 - 3.85 (m, 2H), 3.75 (d, J = 5.9 Hz, 2H), 1.85 (s, 3H). 2-

[0115] 2 (5 mg) was dissolved in 0.7 mL DMSO-d6. Then, EtgN (50 yL) was added, and the mixture was stirred vigorously overnight at RT. Then, the reaction was subjected to high vacuum to remove traces of EtgN and was analysed by 1 H-NMR.

[0116] 1H NMR (500 MHz, DMSO-d6) δ 10.26 (d, J = 2.4 Hz, 1 H), 9.96 (d, J = 2.5 Hz, 1 H), 8.17 (t, J = 6.0 Hz, 1 H), 6.68 (d, J = 2.8 Hz, 1 H), 6.27 (d, J = 2.4 Hz, 1 H), 3.76 (d, J = 5.2 Hz, 2H), 1.85 (s, 3H).

[0117] In situ synthesis of N-; 2 (10 mg, 30 pmol) was dissolved in 1 mL buffer (100 mM Na2HPO4pH 7.5, 150 mM NaCI). Then, 700 mg TsNa (approx. 50 equiv.) was added and the reaction was stirred vigorously for 2 h at RT. The reaction mixture was centrifuged and the product was isolated by RP-HPLC on a C18 column with a gradient of 10-80% MeCN / H2O (0.1% TFA) over 20 min. The geometry of the vinyl was trans.

[0118] In situ synthesis of A / -acetvl-qlvcine-TDAE from elimination

[0119] The elimination product (20 mg, 75 pmol) was dissolved in 1 mL buffer (100 mM Na2HPO4pH 7.5, 150 mM NaCI). Then, 700 mg TsNa (approx. 50 equiv.) was added and the reaction was stirred vigorously for 2 h at RT. Then, the reaction mixture was spun down and the product was isolated by RP-HPLC on a C18 column with a gradient 10-80% MeCN / H2O (0.1 % TFA) over 20 min. The geometry of the double bond was trans.

[0120] 1H NMR (500 MHz, DMSO-d6) δ 10.74 -10.60 (m, 1H), 10.35 -10.20 (m, 1 H), 8.16 (t, J = 6.0 Hz, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.57 (dd, J = 15.0, 1.6 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.01 (dd, J = 15.0, 1.7 Hz, 1H), 3.75 (d, J = 5.9 Hz, 2H), 2.42 (s, 3H), 1.84 (s, 3H).

[0121] TDAE conjugate 3 (3 mg) was dissolved in 150 pL DMSO. Then, 1.35 mL of phosphate buffer (100 mM Na2HPO4pH 7.5, 150 mM NaCI) was added whilst stirring. A stock solution of A / -acetyl-L-cysteine (40 mg / mL) was prepared and 36 pL (1 equiv.) added. The reaction was stirred at 30 °C for 45 min, and the reaction was analysed by LCMS. The product was purified by RP-HPLC on a C18 column with a gradient 5-50% MeCN / FkO (0.1 % TFA) over 20 min. The geometry of the vinyl was trans.

[0122] 1 H NMR (500 MHz, DMSO-d6) δ 9.92 -9.82 (m, 1 H), 9.81 -9.71 (m, 1 H), 8.28 (d, J = 8.1 Hz, 1 H), 8.07 (d, J = 6.0 Hz, 1 H), 7.41 (dd, J = 15.1 , 1.4 Hz, 1 H), 5.92 (d, J = 15.1 Hz, 1 H), 4.38 (ddt, J = 6.6, 4.7, 3.2 Hz, 1 H), 3.68 (d, J = 5.9 Hz, 1 H), 3.19 (dd, J = 13.5, 4.7 Hz, 1 H), 3.02 (dd, J = 13.5, 8.9 Hz, 1 H), 2.47 (s, 1 H), 1 .78 (s, 3H), 1 .78 (s, 3H).

[0123] Preparation of Ub-1-74and Ub-1-75hydrazides:

[0124] Ubi-74 or Ubi-75 were expressed as a fusion with the Mxe-intein and a chitin-binding tag in BL21 (DE3) cells from the pTXB1 vector (NEB). The clarified lysate was incubated with chitin resin (NEB) for 1 h and washed with lysis / wash buffer (50 mM Na2HPO4pH 2, 150 mM NaCI, 2 mM EDTA). Then, the chitin resin was transferred to a bottle with cleavage buffer (50 mM NaOAc pH 5, 100 mM MESNA) and incubated at 4 °C for 2 days on a roller. The resin was filtered off, hydrazine monohydrate (0.5 mL, 100 mM final concentration) was added, and the filtrate was stirred at RT for 1 hour. The pH was then adjusted to 4 and diluted 5-fold, after which the filtrate was loaded onto a 5 mL HiTrap S column, followed by a gradient 0-100% B / A over 20 min (Buffer A = 50 mM NaOAc pH 4, buffer B = 50 mM NaOAc pH 4, 1 M NaCI). The fractions were confirmed by LCMS and the pH was adjusted to 3 for the next step with 4 M HCL

[0125] LCMS: Theoretical mass of Ubi-74-NHNH2 = 8464 Da; observed mass = 8463 Da. Theoretical mass of Ubi-75-NHNH2 = 8521 Da; observed mass = 8520 Da.

[0126] Preparation of Ubi-74 and Ubi-75 TDAE conjugates:

[0127] The combined fractions were concentrated to 8 mL and split into 4 aliquots of 2 mL each. To each of them, the dibromo anhydride (200 equiv., dissolved in 1 mL MeCN) was added under vigorous stirring at room temperature. The centrifuged solution was then injected into a preparative C4 HPLC column (gradient 10-80% MeCN / H2O, 0.1 % TFA over 30 min) for purification and the Ub adducts were freeze-dried.

[0128] LCMS: Theoretical mass of Ubi-74 dibromo adduct = 8678.64 Da; observed mass = 8677 Da. Theoretical mass of HBr elimination = 8597.74 Da; observed mass = 8596 Da. Theoretical mass of Ubi-75 dibromo adduct = 8735.7 Da; observed mass = 8734 Da. Theoretical mass of HBr elimination product = 8654.79; observed mass = 8653 Da. The freeze-dried dibromo compounds were dissolved in 200 pL of a buffer containing 6 M GnCI and 50 mM phosphate at pH 7.5. Then, 1 .8 mL of a buffer containing 100 mM phosphate and 150 mM NaCI was added. Then, solid TsNa (2000 equiv.) was added. The reaction was vigorously stirred at RT for 2 h. Then, 1 mL MQ water was added and the solution was vortexed to allow the total dissolution of TsNa. The solution was centrifuged before purification of the Ub-TDAE adducts by preparative HPLC.

[0129] E2~Ub ABP assembly:

[0130] Recombinant E2 enzyme (UBE2D3 (C21S, 107S, C111S) or UBE2L3 (0175, C137S)) at 20 mg / ml was mixed with an equimolar quantity of Ub-TDAE conjugate and incubated for 2 hours. Product formation and almost complete consumption of Ub-TDAE were confirmed by LCMS. The crude probe mixture was purified by size exclusion chromatography using an AKTA system and an S75 16 / 600 column at 1 mL / min. The running buffer was 100 mM Na2HPO4pH 7.5, and 50 mM NaCI. The fractions were confirmed by SDS-PAGE using a precast 4-12% Bis-Tris gel and visualized by Coomassie staining. Purified ABP was then concentrated to 1.5-4.0 mg / mL with an Amicon Ultra centrifugal filter unit, snap-frozen, and stored at -80 °C in aliquots.

[0131] LCMS: Theoretical mass of Representative mass of UBE2D3~Ubi-75E2~Ub ABP = 27395.1 Da; observed mass = 27390 Da. Theoretical mass of UBE2L3~Ubi-75E2~Ub ABP = 28716.77 Da; observed mass = 28711 Da.

[0132] E3 expression and purification:

[0133] Recombinant E3s were prepared as previously described. In summary, the GST-tagged E3s were recombinantly expressed in BL21 (DE3) cells, purified by affinity chromatography with glutathione sepharose, eluted with GSH, and then further purified by size exclusion chromatography (S200 column and a buffer containing 50 mM Tris and 150 mM NaCI and 1 mM TCEP. The fractions were pooled and concentrated to reach a stock concentration of 3-5 mg / mL. ABP assays:

[0134] In a typical experiment, the E3 was used at 3 pM and the probe at 12 pM. Solutions were degassed by purging with argon for 30 min. Reactions were prepared using a 10X buffer (1 M HEPES pH 7.5, 1 .5 M NaCI, 500 pM TCEP) which was diluted to 1 X by the addition of H2O E3 and ABP. Solutions were degassed by purging with argon for 30 min. Reactions were incubated at the indicated temperature. Samples were taken at the indicated time points and quenched by the addition of 4X LDS (lithium dodecyl sulphate) sample buffer (Thermo Scientific) supplemented with 5% BME. Samples were then resolved by SDS-PAGE with a precast 4-12% Bis-Tris gel (Thermo Scientific) and visualized by Coomassie staining.

[0135] Generality of the Methodology

[0136] To demonstrate the generality of the methodology, ubiquitin-like proteins (Ubls) were employed for X and the E2 conjugating enzyme that is cognate for the respective Ubl was employed for Y.

[0137] The Ubl small ubiquitin-like modifier (SUMO) exists as more than three paralogues (SUMO1-3) that specifically partner with the E2 conjugating enzyme Ubc9 (also known as UBE2I). The ABP molecule comprising SUMO and its E2 conjugating enzyme Ubc9 was prepared by reacting the C-terminal TDAE conjugate of SUMO1 or SUMO2 with the E2 Ubc9 in pH 7.5-8 aqueous buffer.

[0138] The different species were analysed by Coomassie stained SDS-PAGE gel, which confirmed conjugation of species (see Fig. 4 A and B). Samples were also analysed by LC-MS. The chromatogram was monitored at 214 nm. Theoretical mass = 32104.1 Da; found = 32099 Da. A species with mass of 30359 was also observed, which corresponds to inadvertent cleavage of the N-terminal purification tag on Ubc9 due to protease contamination.

[0139] HEK293 lysate was treated with the ABPs for 4 h at 30 °C. Samples were resolved by SDS-PAGE and transferred to membrane and probed with anti SAE2 antibody. SDS- PAGE confirmed that the SUMO E1 activating enzyme subunit containing the catalytic cysteine (SAE2) had been labelled with SUMO ABP. Another Ubl is interferon-stimulated gene 15 (ISG15), where the cognate E2 conjugating enzyme in UBE2L6 (also known as UbcH8). A probe molecule of formula (I) was also synthesised, where X is a N-terminally truncated form of ISG15 (ISG15CTD) and Y is UbcH8.

[0140] The different species were analysed by Coomassie stained SDS-PAGE gel, which confirmed conjugation of species (see Fig. 5 A and B). Samples were also analysed by LC-MS. The chromatogram was monitored at 214 nm. Theoretical mass = 29792.8 Da; found = 29788 Da.

[0141] HERC5 WT and the active cysteine mutant C944Awere incubated with ABP in biological buffer for 4 h at 30 °C. Coomassie stained SDS-PAGE confirmed that only the WT was labelled, despite the protein containing multiple non-catalytic cysteines. Thus, the E2~ISG15CTDABPS undergo activity-dependent covalent labelling of the catalytic HECT domain from the ISG15 E3 HERC5.

Claims

CLAIMS:1 . A compound corresponding to formula (I):wherein X and Y are each independently a biological molecule comprising one or more selected from proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones;Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

2. The compound of claim 1 , wherein Y is an E2 conjugating enzyme (E2).

3. The compound of claim 2, wherein the E2 is selected from UBE2D1 , UBE2D2, UBE2D3 or UBE2D3.

4. A compound corresponding to formula (II):wherein X is a biological molecule comprising one or more selected from proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones;Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl;each R2is independently selected from the group consisting of H, nitrile or halo; and LG is a leaving group.

5. The compound of claim 4, wherein LG is selected from the group consisting of chloro, bromo, or iodo.

6. The compound of claim 4, wherein LG corresponds to formula (III):wherein R3is selected from the group consisting of C1-6alkyl; C1-6haloalkyl; phenyl; and naphthyl, optionally substituted one or more times with C1-6alkyl, C1-6alkoxy, halo, amino, alkylamino, or nitro.

7. The compound of claim 6, wherein R3is selected from the group consisting of methyl, trifluoromethyl, 4-methylphenyl, 4-nitrophenyl, 4-bromophenyl, 2,2,2-trifluoroethyl, and 5-(dimethylamino)naphthalyl.

8. The compound of any one of claims 1 to 7, wherein X is ubiquitin or ubiquitin- like protein.

9. The compound of any one of claims 1 to 8, wherein Z is -N(R1)-.

10. The compound of any one of claims 1 to 9, wherein R1is H.11 . The compound of any one of claims 1 to 10, wherein R2is H.

12. A method for preparing a compound as defined in claim 1 , the method comprising contacting a biological molecule comprising a thiol moiety with a compound of formula (II):wherein X is a biological molecule comprising one or more selected from proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones;Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and LG is a leaving group.

13. The method of claim 12, wherein LG is as defined in any one of claims 5 to 7.

14. The method of claim 12 or claim 13, wherein the thiol moiety is a cysteine residue.

15. The method of claim 14, wherein the cysteine residue is naturally occurring or wherein the cysteine residue has been introduced to the biological molecule.

16. A method of detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, the method comprising providing a first compound of formula (I):wherein X and Y are each independently a biological molecule comprising one or more selected from proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones;Z is -O- or -N(R1)-;each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; each R2is independently selected from the group consisting of H, nitrile and halo; and contacting the compound of formula (I) with one or more biological molecules, such that any of the one or more biological molecules with transthiolation activity are able to react with the compound of formula (I) to form a conjugate; and detecting the formation, or lack thereof, of conjugates.

17. The method of claim 16, wherein Y is as defined in claim 2 or claim 3.

18. The method of claim 16 or claim 17, wherein the one or more biological molecules is one or more ubiquitin-activating enzymes (E1s) or one or more ubiquitin ligases (E3s).

19. The method of any one of claims 16 to 18, wherein the detecting the formation, or lack thereof, of the one or more conjugates is carried out by a method selected from the group consisting of gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), fluorescence polarization, fluorescence resonance energy transfer, magnetic separation, nuclear magnetic resonance (NMR) spectroscopy, immunological separation, gel filtration chromatography, affinity chromatography, column chromatography, displacement chromatography, electro-chromatography, gas chromatography (GC), high- performance liquid chromatography (HPLC), liquid-chromatography mass- spectrometry (LCMS), ion chromatography, micellar electrokinetic chromatography, thin-layer chromatography (TLC), centrifugation, adhesion, and flow cytometry.

20. The method of claim 19, wherein the detecting the formation, or lack thereof, of the one or more conjugates is carried out by SDS polyacrylamide gel electrophoresis.21 . The method of any one of claims 12 to 20, wherein X is ubiquitin or ubiquitin- like protein.

22. The method of any one of claims 12 to 21 , wherein Z is -N(R1)-.

23. The method of any one of claims 12 to 22, wherein R1is H.

24. The method of any one of claims 12 to 23, wherein R2is H.

25. Use of a compound corresponding to formula (I) for detecting interaction or lack thereof between a compound of formula (I) and one or more biological molecules, wherein formula (I) is:wherein X and Y are each independently a biological molecule comprising one or more selected from proteins, peptides, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones;Z is -O- or -N(R1)-; each R1is independently selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, and C2-6haloalkenyl; and each R2is independently selected from the group consisting of H, nitrile and halo.

26. The use of claim 25, wherein Y is as defined in claim 2 or claim 3.

27. The use of claim 25 or claim 26, wherein:X is ubiquitin or ubiquitin-like protein;Z is -N(R1)-;R1is H; and / orR2is H.

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