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  • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
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  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6806—Determination of free amino acids
  • G01N33/6812—Assays for specific amino acids
  • G01N33/6815—Assays for specific amino acids containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
• • A—HUMAN NECESSITIES
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
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  • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
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  • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
  • C12Y108/04—Oxidoreductases acting on sulfur groups as donors (1.8) with a disulfide as acceptor (1.8.4)
  • C12Y108/04013—L-Methionine (S)-S-oxide reductase (1.8.4.13)
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  • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
  • C12Y108/04—Oxidoreductases acting on sulfur groups as donors (1.8) with a disulfide as acceptor (1.8.4)
  • C12Y108/04014—L-Methionine (R)-S-oxide reductase (1.8.4.14)
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  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
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  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

• Cysteine bioconjugation strategies typically exploit the intrinsically high nucleophilicity of the thiol/thiolate side chain, including elegant methods based on electrophilic warheads such as maleimides and alkyl and aryl halides, 26-30 transition metal-mediated bioconjugation, 31 and cysteine-to-dehydroalanine conversion.28 ' 29 ' 32 ' 33
• methionine is among the most hydrophobic and rare amino acids, and taken together with the fact that the majority of methionine residues are buried within interior protein cores, 1 ' 2 surface- accessible methionines are limited and offer a potentially valuable handle for site-selective protein modification using naturally-occurring amino acid side chains.
• post-translational modifications of methionine including by oxidation and/or metal binding, 3 ' 34 ' 35 are emerging as critical nodes in signaling pathways that control function at the cell and organism level.
• a major chemical challenge in developing a selective methionine modification reaction is its relatively weak nucleophilicity, which precludes the traditional approach of identifying an appropriate methionine-specific electrophilic partner for its acid-base bioconjugation in the presence of competing amino acids that possess stronger nucleophilicity such as cysteine, lysine,
• the methods of the present invention are referred to as Redox Activated Chemical Tagging (ReACT).
• the invention provides methods for the chemoselective conjugation to a target molecule comprising one or more thioether moieties. These methods comprise reacting an N-transfer oxidant with a thioether substrate in a redox reaction in an aqueous environment to form a conjugation product.
• the N-transfer oxidant comprises a reactive oxaziridine group, which reacts with at least one of the one or more thioether moieties on the target molecule, and the conjugation product comprises a resultant sulfimide on the target molecule.
• an N-transfer oxidant is reacted with a thioether moiety on a target molecule in an aqueous environment.
• Suitable N-transfer oxidants comprise an N-halide bond, a N-0 bond, or an N-S bond, and preferably comprise a oxaziridine group.
• Suitable substrates comprise a thioether.
• the oxidant and thioether are represented
• Ri comprises a carboxyl or amide group bound to the indicated N, and further comprises a functional group (e.g., a functional group that can be reacted in a click reaction, for example, but not limited to, an azide or alkyne group), which can be further reacted to form a linker to a payload molecule comprising a corresponding functional group (e.g.
• a functional group that can be reacted in a click reaction for example, but not limited to, payload molecules modified to comprise an azide or alkyne group.
• X is O or NH
• R 8 is Ci_ 6 alkyl optionally substituted with C 2 -6 alkynyl, C 2 -6 alkynyloxy, or -N3.
• R 2 is H, halogen, Ci_6 alkyl, Ci_6 haloalkyl, Ci_6 alkoxy, or Ci_6 haloalkoxy
• R3 is phenyl or heteroaryl, wherein phenyl or heteroaryl are optionally substituted with one or more (e.g.
• Ri is -C(0)XR 8
• X is O or NH
• Rg is Ci_6 alkyl optionally substituted with C 2 -6 alkynyl, C 2 -6 alkynyloxy, or -N 3
• R 2 is H, Ci_6 alkyl, or Ci_6 haloalkyl
• R 3 is phenyl, for compounds of formula I.
• R4 is Ci_6 alkyl and R5 is substituted Ci_6 alkyl such that R4-S-R5 is an amino acid residue of a protein or polypeptide, preferably wherein R4-S-R5 is a methionine residue of protein or polypeptide.
• R4-S-R5 represents a methionine thioether of a protein or polypeptide target molecule.
• proteins or polypeptides include, without limitation, a therapeutic protein or polypeptide, including an antibody or antibody fragment.
• the protein or polypeptide includes one or more additional methionine residues that may also react with the N- transfer oxidant in the reaction with compounds of formula I.
• the compound of formula II is a protein or polypeptide having one or more methionine residues, wherein one or more of the methionine residues reacts with the N-transfer oxidant.
• R 4 is methyl and R 5 is -CH 2 CH 2 CH(NHR 6 )C(0)R 7 , wherein R 6 is -H or NHR 6 forms a peptide bond, and R 7 is -OH, or C(0)R 7 forms a peptide bond, provided that at least one of NHR 6 and C(0)R 7 forms a peptide bond.
• the compound of formula II is a compound of formula Ila
• NH-R 6 and C(0)-R 7 are peptide bonds within a protein or peptide, or R 6 is -H and C(0)-R 7 is a peptide bond within a protein or peptide, or R 7 is -OH and NHR 6 is a peptide bond within a protein or peptide.
• Ri comprises a functional group, wherein said functional group can be further reacted to form a linker to a payload molecule, such as an active moiety.
• R a linker to a payload molecule, such as an active moiety.
• Rg is Ci_6 alkyl optionally substituted with C2-6 alkynyl, C2-6 alkynyloxy, or -N 3 ;
• R2 is H, halogen, Ci_6 alkyl, Ci_6 haloalkyl, Ci_6 alkoxy, or Ci_6 haloalkoxy, and
• R 3 is phenyl or heteroaryl, wherein phenyl or heteroaryl are optionally substituted with one or more (e.g.
• substituents independently selected from the group consisting of halogen, Ci_6 alkyl, Ci_6 haloalkyl, Ci_6 alkoxy, and Ci_6 haloalkoxy.
• R2 is H; and R 3 is phenyl.
• the compound of formula I is a compound of formula la
• X is NH
• R 6 and R 7 are as defined for compounds of formula II and X and Rg are as defined for compounds of formula I.
• X is NH.
• the compound of formula III is a compound of formula Ilia
• P represents a polypeptide or protein linked to the indicated nitrogen via a sulfimide bond with the sulfur of methionine, wherein the polypeptide or protein has n sulfimide modified methionine residues, where n is 1 or more, including 1-20, 1-15, 1-10, 1-5, 1, 2, 3, 4 or 5, and X and Rg are as defined for compounds of formula I.
• X is NH.
• a target molecule conjugate comprising a target molecule comprising at least one sulfimide modified methionine residue bound to a linker, wherein said linker is bound to a payload molecule.
• the payload molecule is an active moiety.
• the target molecule is a protein or polypeptide and the conjugate is a protein or polypeptide conjugate of formula IV
• R 6 and R 7 are as defined in formula II, X is O or NH, L is a linker moiety and Rg is a payload molecule.
• the payload molecule is an active moiety.
• X is NH.
• L is a linker resulting from the click reaction of a compound of formula III (including a compound of formula Ilia) and a suitably modified payload molecule.
• Suitably modified payload molecules contain substituents reactive in click reactions, for example, but not limited to, payload molecules modified to comprise an azide or alkyne group (see e.g., Fig. 3).
• the compound of formula IV is a compound of formula IVa
• P represents a polypeptide or protein linked to the indicated nitrogen via a sulfimide bond with the sulfur of methionine, wherein the polypeptide or protein has n sulfimide modified methionine residues, where n is 1 or more, including 1-20, 1-15, 1-10, 1-5, 1, 2, 3, 4 or 5,
• X is O or NH
• L and Rg are as defined for a compound of formula IV.
• X is NH.
• L is a linker resulting from the click reaction of a compound of formula III (including a compound of formula Ilia) and a suitably modified payload molecule.
• L comprises a triazole or isoxazole ring linking group.
• the compound of formula IV is a compound of formula IVb
• Li comprises a triazole or isoxazole ring linking group.
• P is a polypeptide or protein, such as an enzyme, an antigenic protein, a chemokine, a cytokine, a cellular receptor, a ligand for a cellular receptor, or an antibody or active fragment thereof.
• an enzyme an antigenic protein, a chemokine, a cytokine, a cellular receptor, a ligand for a cellular receptor, or an antibody or active fragment thereof.
• the antibody or active fragment thereof comprises a modification of the wild type antibody by introducing one or more accessible methionine residues.
• the invention provides a redox-activated chemical tagging (ReACT) method for methionine-based substrate functionalization, comprising contacting a methionine- containing substrate with an oxaziridine in an aqueous environment wherein the oxaziridine directly functionalizes the substrate by converting the methionine of a substrate to the corresponding sulfimide conjugation product.
• ReACT redox-activated chemical tagging
• the invention provides reacting a protein, such as an antibody or active fragment thereof, with a compound of formula I (including a compound of formula la) to provide a compound formula III (including a compound of formula Ilia). In some embodiments, the invention further provides reacting a compound of formula III (including a compound of formula Ilia) to form a compound of formula IV (including a compound of formula IVa or IVb). In some embodiments, the invention further provides reacting a compound of formula Ilia to form a compound of formula IVa or IVb, preferably wherein P is an antibody or active fragment thereof.
• the substrate is a protein
• the method results in modification of the protein, with applications in synthesis and characterization of antibody-drug conjugates and related biologic therapeutics and imaging agents, chemoproteomics and inhibitor design, as well as modifications to study and improve upon protein function, including solubility, stability, and metabolism and pharmacokinetics.
• the subject methods and ReACT methods can be combined with stable isotope labeling with amino acid in cell culture (SILAC) or isotope coded affinity tag (ICAT) for quantitative proteomics analysis of methionine function in vivo and in vitro by mass spectrometry, with application including but not limited to quantitative analysis of methionine reactivity, quantitative analysis of oxidative-sensitive methionine, quantitative analysis of stress sensitive methionine and quantitative analysis of methionine sulfoxide reductase substrates.
• the invention provides compounds adapted for use in a redox-activated chemical tagging (ReACT) method.
• the invention provides a compound of formula I (including a compound of formula la); a compound of formula II (including a compound of formula Ila); or a compound of formula III (including a compound of formula Ilia).
• the invention provides a polypeptide or protein conjugate which has the structure of formula IV (including a structure of formula IVa or IVb), wherein P is a polypeptide or protein such as an enzyme, an antigenic protein, a chemokine, a cytokine, a cellular receptor, or an antibody or active fragment thereof.
• the invention encompasses all combination of the particular embodiments recited herein, as if each combination had been laboriously recited.
• Fig 1A-D The ReACT strategy for chemoselective methionine bioconjugation.
• Fig 2A-B The ReACT strategy for protein functionalization.
• A General two-step procedure for methionine- specific protein functionalization a combination of ReACT and click reactions.
• B Redox conjugation of a CaM model protein with various oxaziridine (Ox) compounds.
• Fig 3 Functionalization of ReACT labeled calmodulin protein with various payloads using click reaction. Desthiobiotin, Cy3 and PEG payloads comprising reactive groups suitable for conjugation using click chemistry.
• Therapeutic proteins functionalization based on methionine bioconjugation such as therapeutic protein PEGylation, antibody-drug conjugates, protein labeling for imaging and diagnosis, as well as other protein post translational modifications.
• Therapeutic polypeptides functionalization based on methionine bioconjugation such as polypeptide PEGylation, polypeptide-drug conjugates and other polypeptide post- translational modifications.
• the present invention provides a unique and general redox-based approach to chemoselective methionine conjugation that complements the wealth of acid-base conjugation methods for modification of more nucleophilic amino acids such as cysteine, lysine, and serine.
• Three major utility directions of this invention are listed as follow:
• One utility of the present invention is to provide for the functionalization of proteins and polypeptides, such as antibodies and their fragments, as well as other therapeutic proteins, using naturally-occurring methionine residues that are native or readily introduced by chemical modification or by standard site-directed mutagenesis. This procedure is simple and
• poly(alkyl oxide) e.g. "PEG”
• fluorophores e.g. "FecA”
• other payloads or tracers e.g. "FecA”
• conjugate two polypeptides together e.g., an antibody/protein conjugate
• the present invention provides for installation of various payloads as mentioned above onto other biomolecules, including but not limit to, DNA, RNA, lipid and sugar by introduction a thioether function group. This method allows functionalization of any thioether containing biomolecules with a simple and straightforward procedure.
• the selectivity of the oxaziridine as the N-transfer oxidant offers a chemical platform for identifying and studying functional methionines in whole proteomes, and function protein using methionine as key residues, providing a vehicle for therapeutic interventions based on reactive methionine activation and/or inhibition.
• the present invention can provide several benefits as compared to other naturally- occurring amino acids such as cysteine, lysine and tyrosine based bioconjugation methods.
• the present invention provides highly selective, rapid, and robust methionine labeling methodology that is operable under a range of biocompatible reaction conditions using redox based reactivity without using electrophiles to label protein, thus avoiding a selectivity issue, and a resulting inconsistency of labeling in a protein drug.
• the present invention can permit installation of various payloads onto proteins at well-defined positions and with excellent pay load- target molecule conjugation efficiency due to the extremely high reactivity of the oxaziridine group with the thioether.
• the protein functionalization of the present invention can be completed within 20 min at neutral pH and under biocompatible conditions.
• the reactivity kinetics of our method is much faster than traditional protein modification methods. This provides a significantly simplified the protein functionalization procedure.
• the bioconjugation linkages described herein are stable for at least 14 days in the presence of 100% FBS, which is beneficial for in vivo applications.
• the protein functionalization is also stable in an extracellular environment. More importantly, under strong thiol reducing conditions such as are found intracellularly, the linkage can be cleaved to release an attached active moiety (e.g. a therapeutic) within a cell.
• the present invention also may provide significant advantages over non-natural amino acid based bioconjugation methods.
• the present methods enable the direct functionalization of a protein of interest at directed methionine residues without engineering the non-natural functionalities into a protein through such non-natural amino acids.
• a hydrocarbyl group is a substituted or unsubstituted, straight-chain, branched or cyclic alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group which comprises 1-15 carbon atoms and optionally includes one or more heteroatoms in its carbon skeleton.
• heteroatom as used herein generally means any atom other than carbon or hydrogen.
• Preferred heteroatoms include oxygen (O), phosphorus (P), sulfur (S), nitrogen (N), and halogens
• preferred heteroatom functional groups are haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide, isocyanate, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.
• alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which is fully saturated, having the number of carbon atoms designated (i.e. Ci -8 means one to eight carbons).
• alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.
• alkenyl by itself or as part of another substituent, means a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-8 means two to eight carbons) and one or more double bonds.
• alkenyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl) and higher homologs and isomers thereof.
• alkynyl by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-8 means two to eight carbons) and one or more triple bonds.
• alkynyl groups include ethynyl, 1- and 3- propynyl, 3-butynyl and higher homologs and isomers thereof.
• alkylene by itself or as part of another substituent means a divalent radical derived from alkyl, as exemplified by -CH 2 -CH 2 -CH 2 -CH 2 -.
• an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the invention.
• a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
• alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
• haloalkoxy refers to a haloalkyl group attached to the remainder of the molecule via an oxygen atom.
• heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, P, Si and S, wherein the nitrogen, sulfur, and phosphorous atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
• the heteroatom(s) O, N, P and S may be placed at any interior position of the heteroalkyl group.
• the heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.
• Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and -CH 2 -0-Si(CH 3 ) 3 .
• heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 - S-CH 2 -CH 2 -NH-CH 2 -.
• heteroalkylene groups heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.
• cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Accordingly, a cycloalkyl group has the number of carbon atoms designated (i.e., C 3 _8 means three to eight carbons) and may also have one or two double bonds.
• C 3 _8 means three to eight carbons
• heterocycloalkyl group consists of the number of carbon atoms designated and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
• heterocycloalkyl examples include l-(l,2,5,6-tetrahydropyrid- yl), 1-piperidinyl, 2-piperidinyl, 3- piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
• halo and "halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
• terms such as “haloalkyl,” are meant to include alkyl substituted with halogen atoms, which can be the same or different, in a number ranging from one to (2m'+l), where m' is the total number of carbon atoms in the alkyl group.
• Ci- 4 haloalkyl is meant to include
• haloalkyl includes monohaloalkyl (alkyl substituted with one halogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms in a number ranging from two to (2m'+l) halogen atoms, where m' is the total number of carbon atoms in the alkyl group).
• perhaloalkyl means, unless otherwise stated, alkyl substituted with (2m'+l) halogen atoms, where m' is the total number of carbon atoms in the alkyl group.
• Ci- 4 perhaloalkyl is meant to include trifluoromethyl, pentachloroethyl, l,l,l-trifluoro-2-bromo-2-chloroethyl and the like.
• acyl refers to those groups derived from an organic acid by removal of the hydroxy portion of the acid. Accordingly, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.
• aryl means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
• aryl groups include phenyl, 1- naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.
• heteroaryl refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom are optionally quaternized.
• a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
• heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl and 6-
• aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
• arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like).
• alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
• an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naph
• R', R" and R' each independently refer to hydrogen, unsubstituted Ci-8 alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-Ci-4 alkyl groups.
• R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring.
• -NR'R is meant to include 1-pyrrolidinyl and 4- morpholinyl.
• an alkyl or heteroalkyl group will have from zero to three substituents, with those groups having two or fewer substituents being preferred in the invention. More preferably, an alkyl or heteroalkyl radical will be unsubstituted or monosubstituted. Most preferably, an alkyl or heteroalkyl radical will be unsubstituted. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups such as trihaloalkyl (e.g., -CF 3 and -CH 2 CF 3 ).
• the aryl group When the aryl group is 1,2,3,4-tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted C 3 -7 spirocycloalkyl group.
• the C 3 -7 spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".
• an aryl or heteroaryl group will have from zero to three substituents, with those groups having two or fewer substituents being preferred in the invention.
• an aryl or heteroaryl group will be unsubstituted or monosubstituted.
• an aryl or heteroaryl group will be unsubstituted.
• Preferred substituents for aryl and heteroaryl groups are selected from: halogen, -OR', - OC(0)R, -NR'R", -SR', -R, -CN, -N0 2 , -C0 2 R, -CONR'R", -C(0)R',-OC(0)NR'R", - NR"C(0)R, -S(0)R, -S0 2 R, -S0 2 NR'R", -NR"S0 2 R, -N 3 , -CH(Ph) 2 , Ci_ 4 perfluoroalkoxy and Ci_ 4 perfluoroalkyl, where R' and R" are as defined above.
• substituents are selected from: halogen, -OR, -OC(0)R', -NR'R", -R, -CN, -N0 2 , -C0 2 R, -CONR'R", - NR"C(0)R, -S0 2 R, -S0 2 NR'R", -NR"S0 2 R, Ci_ 4 perfluoroalkoxy and Ci_ 4 perfluoroalkyl.
• the substituent -C0 2 H includes bioisosteric replacements therefore; see, e.g., The Practice of Medicinal Chemistry; Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.
• Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH 2 )q-U-, wherein T and U are independently -NH-, -0-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CH 2 -, -0-, -NH-, -S-, -S(0 , -S(0) 2 -, -S(0) 2 NR- or a single bond, and r is an integer of from 1 to 3.
• One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH 2 )s-X- (CH 2 )t- -, where s and t are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, - S(O)-, -S(0) 2 -, or -S(0) 2 NR'-.
• the substituent R' in -NR'- and -S(0) 2 NR'- is selected from hydrogen or unsubstituted Ci_ 6 alkyl.
• applicable substituents are independently substituted or unsubstituted heteroatom, substituted or unsubstituted, optionally heteroatom Ci_6 alkyl, substituted or unsubstituted, optionally heteroatom C 2 _ 6 alkenyl, substituted or unsubstituted, optionally heteroatom C 2 _ 6 alkynyl, or substituted or unsubstituted, optionally heteroatom C 6 -i4 aryl, wherein each heteroatom is independently oxygen, phosphorus, sulfur or nitrogen.
• applicable substituents are independently aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl, alkyloxy, alkyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido, carboxyl, cyanyl, ester, halo, haloformyl, hydroperoxyl, hydroxyl, imine, isocyanide, isocyanate, N-tert-butoxycarbonyl, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, sulfhydryl, thiol, thiocyanyl, trifluoromethyl or trifluromethyl ether (OCF 3 ).
• peptide refers to at least two amino acids joined by peptide bonds.
• a "polypeptide” refers to a short sequence of amino acids (less than 50), where the amino acids are connected to each other by peptide bonds.
• a peptide or polypeptide may occur free or bound to another moiety, such as a macromolecule, lipid, oligo- or polysaccharide, and/or a polypeptide. Where a peptide is incorporated into a polypeptide chain, the term “peptide” may still be used to refer specifically to the short sequence of amino acids.
• a peptide or polypeptide may be connected to another moiety by way of a peptide bond or some other type of linkage.
• a polypeptide is more than two amino acids in length and generally less than about 25 amino acids in length.
• the terms "peptide” and “oligopeptide” may be used interchangeably.
• Protein generally refers to the sequence of amino acids comprising a polypeptide chain and that is greater than 50 amino acids in length. Protein may also refer to a three dimensional structure of the polypeptide. “Denatured protein” refers to a partially denatured polypeptide, having some residual three dimensional structure or, alternatively, to an essentially random three dimensional structure, i.e., totally denatured.
• the invention encompasses reagents of, and methods using, polypeptide variants, e.g., involving glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalent and non-covalently bound cofactors, oxidized variants, and the like.
• polypeptide variants e.g., involving glycosylation, phosphorylation, sulfation, disulfide bond formation, deamidation, isomerization, cleavage points in signal or leader sequence processing, covalent and non-covalently bound cofactors, oxidized variants, and the like.
• disulfide linked proteins is described (see, e.g., Woycechowsky and Raines (2000) Curr. Opin. Chem. Biol. 4:533-539; Creighton, et al. (1995) Trends Biotechnol. 13: 18-23
• antibody refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g.,
• antibody includes antigen-binding portions, i.e., "antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
• Antigen binding sites e.g., fragments, subs
• cytokine as used herein means any secreted polypeptide that affects the functions of cells and is a molecule which modulates interactions between cells in the immune, inflammatory or hematopoietic response.
• a cytokine includes, but is not limited to, monokines and lymphokines regardless of which cells produce them. Examples of cytokines include, but are not limited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-a) and Tumor Necrosis Factor-beta (TNF-b).
• IL-1 Interleukin-1
• IL-6 Interleukin-6
• IL-8 Interleukin-8
• TNF-a Tumor Necrosis Factor-alpha
• TNF-b Tumor Necrosis Factor-beta
• receptor refers to a molecule, typically composed primarily of protein, that binds to a cognate ligand and that is associated, or derived from, a cell, and usually one or more types of cellular membranes, and has as its main biological function the ability to bind a specific ligand or group of ligands, and, upon ligand binding, to mediate signal transduction, either directly or indirectly, in the cell.
• One type of receptor has three portions or domains, namely, an intracellular domain, an extracellular domain, and a transmembrane domain.
• the receptor typically has a sequence of about 5-25 amino acids at its amino terminus that serve to target the receptor to the proper membrane.
• the extracellular domain binds to one or more ligands
• the transmembrane domain anchors the receptor into the membrane
• the intracellular domain perceives the binding of ligand and transmits a signal to the interior of the cell (the intracellular environment).
• chemokine refers to a member of one of 4 different structural families, comprising over 50 ligands that interact with at least 17 different receptors These chemokine families, named according to the structure of a conserved cysteine-containing motif, are defined by the presence of either a C, a CC, a CXC, or a C'C at their amino terminus of the protein.
• amino acid peptide e.g. as used in describing compounds of formula II (including a compound of formula Ila) refers to the bonds within a protein or polypeptide, wherein the thioether containing substrate includes an amino acid peptide, e.g.
• the thioether is within a methionine residue of a polypeptide or protein.
• the substrate R4-S-R5 represents e.g. a thioether wherein R 4 and R5 are alkyl groups, and the alkyl group of R5 is substituted at one carbon with both the -NHC(0)R, and -C(0)NHR' groups within a polypeptide or protein (e.g. where R is an additional peptide chain or a carboxyl terminus OH and R' is an additional peptide chain or an amino terminus H).
• an alkyl group, e.g. of R5 substituted with an amino acid peptide can be represented as
• substrate R4-S-R5 represent a methionine residue tide
• the substrate may comprise a polypeptide or protein that has additional modifications, or may have additional methionine residues that can be reacted by the methods of the present invention as described herein.
• an "active moiety” is a payload molecule as described in the present invention, wherein said moiety conjugated to e.g. a protein or polypeptide provides some activity.
• the activity of the moiety includes, but is not limited to, a biological activity (e.g. a pharmaceutically active moiety, such as a small molecule pharmaceutical or a biomolecule, such as DNA, RNA, lipid or sugar), a detectable label (e.g. fluorophore, imaging label or the like), a property modifying moiety (e.g.
• PEGylation moiety a sulfhydryl-specific functional moiety such as a maleimide, alkyl or aryl halide, a-haloacyl, or pyridyl disulfides, an amine-specific functional moiety such as a carbodiimide, a non-selective reactive moiety such as a photoaffinity group, an arginine- specific functional moiety such as a glyoxal, etc.
• a sulfhydryl-specific functional moiety such as a maleimide, alkyl or aryl halide, a-haloacyl, or pyridyl disulfides
• an amine-specific functional moiety such as a carbodiimide
• a non-selective reactive moiety such as a photoaffinity group
• an arginine- specific functional moiety such as a glyoxal, etc.
• click chemistry or “click reaction” refers to well-known, selective methods of conjugation, wherein two components comprising a click reactive functional group are reacted to link the two components.
• the modified group further comprises a first click reactive functional group
• the payload molecule is suitably modified to comprise a second click reactive functional group, which is reactive with the first click reactive functional group.
• the click reactive functional group includes, without limitation, an azide group, a nitrone group or an alkyne group.
• click chemistry comprises reaction of an azide group with an alkyne group to form a triazole group linking the two components, or the reaction of a nitrone group with an alkyne group to form an isoxazoline group linking the two components.
• the alkyne group is a dibenzocyclooctyne (DBCO) group or a difluorooctyne (DIFO) group.
• the click chemistry is Copper(I)- catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC) or strain-promoted alkyne-nitrone cycloaddition (SPANC).
• CuAAC Copper(I)- catalyzed azide-alkyne cycloaddition
• SPAAC strain-promoted azide-alkyne cycloaddition
• SPANC strain-promoted alkyne-nitrone cycloaddition
• Fig 1A Left panel: Acid-base conjugation strategies for cysteine-based protein functionalization; Right panel: Redox- Activated Chemical Tagging (ReACT) strategies for methionine-based protein functionalization.
• Oxaziridine (Ox) compounds serve as oxidant- mediated reagents for direct functionalization by converting methionine to the corresponding sulfimide conjugation product.
• SI N-acetyl-L- methionine methyl ester
• ReACT as a method for site- selective methionine conjugation of proteins was evaluated.
• BSA bovine serum albumin
• CuAAC copper-catalyzed azide-alkyne cycloaddition
• ReACT proceeds rapidly and can be completed with a yield > 95% within 1-2 min, with 50% of labeling occurring within the first 5 s following the addition of Ox4 to the protein under standard reaction conditions (Fig. ID).
• Fig. ID This shows that the ReACT method could enable installation of various payloads onto a protein of interest at defined methionine sites, serving as a unique method for functionalization using naturally occurring amino acids (Fig. 2A).
• Fig. 2B The reactivity of various alkyne- and azide-containing oxaziridine probes with CaM model protein was assessed (Fig. 2B).
• Fig 2A General two-step procedure for methionine-specific protein functionalization a combination of ReACT and click reactions.
• Various payloads can be installed through methionine conjugation at a directed position on a given protein.
• B Redox conjugation of a CaM model protein (100 ⁇ ) with various oxaziridine (Ox) compounds (1 mM).
• Ox oxaziridine
• the chemical structures of oxaziridine probes are shown with molecular weight changes ( ⁇ ) listed for the corresponding modifications.
• the deconvoluted MS data of full protein peaks are plotted in the same figure.
• Ox2 labeled protein expected mass 17,564 Da, found 17,565 Da
• Ox4 labeled protein expected mass 17,654 Da, found 17,654 Da
• Ox5 labeled protein expected mass 18,050 Da, found 18,051 Da
• Ox6 labeled protein expected mass 18,059 Da, found 18,060 Da.
• ADCs antibody-drug conjugates
• GFP-Fab anti-green fluorescent protein antibody fragment
• an HEK-293T cell line with a doxycycline (Dox) inducible cell surface GFP expression system was used, where Dox treatment results in expression of GFP localized to the cell surface.
• Dox treatment results in expression of GFP localized to the cell surface.
• Cy3 -labeled GFP-Fab made by ReACT
• excellent co-localization of Cy3 and GFP signals in live HEK-293T cells was observed.
• no Cy3 signal was observed in control cells without Dox addition.
• the intensity of the Cy3 signal was found to be stable for at least 14 days in the presence of 100% fetal bovine serum (FBS).
• ReACT can enable antibody functionalization at directed positions with a wide variety of payloads and simultaneously retain their function for ligand binding.
• ReACT was also applied to a therapeutic conjugate, Herceptin-Fab (Her-Fab). ReACT does not label wildtype Her-Fab owing to its lack of surface accessible methionines. By engineering Her-Fab platforms carrying one or two methionine residues at the C-terminus, ReACT affords quantitative conjugation with one or two redox modifications, respectively.
• DAR drug-to-antibody ratio
• the bioorthogonal azide or alkyne handle introduced by methionine conjugation can be readily functionalized with additional payloads.
• the ADC synthesized by linking monomethyl auristatin E (MMAE) to Her-Fab exhibits a 5-fold increase in toxicity to Her2 -positive BT474 breast cancer cells compared to either wildtype Her-Fab or a mixture of wildtype Her-Fab and free MMAE, demonstrating its utility in a biological context (See Lin et al., Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597-602 (2017), the disclosure of which is hereby incorporated by reference in its entirety).
• ReACT may be used as a unique methionine-targeted warhead for chemoproteomics applications, owing to its high specificity and reactivity, as well as its small size for accessing a broad range of proteins.
• ReACT was applied to probe reactive methionines in the proteome through tandem orthogonal proteolysis - activity based protein profiling (TOP- ABPP). 45
• TOP- ABPP tandem orthogonal proteolysis - activity based protein profiling
• hyper-reactive methionine targets are the hyper-reactive methionine targets, as they can predict sites of methionine-regulated protein function.
• This unbiased ReACT approach not only enables characterization of previously studied redox- sensitive methionines in whole proteome settings, but more importantly identifies new functional methionine sites.
• three hyper-reactive methionines within actin were identified, including Met 44 and Met 46, whose redox activities have been previously shown to play a central role in controlling actin polymerization in living cells. 36"38 With these data validating the ReACT method in hand, it was attempted to identify and characterize new targets with methionine-dependent function.
• enolase a central enzyme in the ancient and conserved metabolic pathway of glycolysis, which is of importance in regulating diseases such as cancer via the Warburg effect. 47 Included is a Met 169 residue that is highly conserved from yeast to mammals (corresponding to Met 171 on yeast enolase 1). Met 169 is close to the enzyme active site and can be oxidized along with other methionine residues in the mammalian protein upon oxidant treatment.
• References include sulfur' s central role in biological processes (52-56), selective protein conjugation methods based on cysteine and their applications (57-64), existing methionine labeling methods with acid-base mechanism (65-67), and reviews on oxaziridine chemistry (68- 69).
• immunoprecipitation assay buffer (RIP A) were labeled with the same protocol used for model protein samples. Samples were subsequently quenched by protein precipitation with cold methanol. The pellets were washed twice with cold methanol, air dried and solubilized with 2% SDS/PBS.
• Oxaziridine labeled protein or proteome samples carrying alkyne handles at the concentration 1 mg/mL in PBS were labeled with the CuAAC reaction.
• the reactions were performed by addition of 1 mM CuSC (lOOx stock in water), 100 ⁇ Tris(benzyltriazolylmethyl)amine (TBTA, lOOx stock in DMSO), 100 ⁇ azide- PEG3-biotin or azide-Cy3 (lOOx stock in DMSO, Click Chemistry Tools) and 2 mM sodium ascorbate (lOOx stock in water).
• the reactions were then agitated for 1 hour at room temperature before quenching with 5 mM disodium bathocuproine disulfonate (BCS, lOOx stock in water).
• BCS disodium bathocuproine disulfonate
• Protein samples carrying azide handles at the concentration 1 mg/mL in PBS were labeled with dibenzocyclooctyne (DBCO) containing compounds using copper-free click reaction.
• DBCO dibenzocyclooctyne
• the reactions were performed by addition of 2-10 equivalents of DBCO-Biotin, DBCO-Cy3, DBCO- PEG-lOkDa or DBCO-MMAE and reacted for 8 hour at room temperature before quenching by protein desalting.
• Her-Fab modifications were analyzed in the positive ion mode on a Synapt G2-Si mass spectrometer that was equipped with an ionKey ESI source and C4 ionKey (150 ⁇ inner diameter, 50 mm length, 300 A pore size, 1.7 ⁇ particle size) and connected in line with an Acquity M-class liquid chromatograph (Waters). Mass spectrometry measurements of GFP-Fab were obtained at the University of California, San Francisco.
• GFP-Fab modifications were analyzed in the positive ion mode on a Xevo G2-XS mass spectrometer equipped with a LockSpray (ESI) source and Acquity Protein BEH C4 column (2.1 mm inner diameter, 50 mm length, 300 A pore size, 1.7 ⁇ particle size) connected in line with an Acquity I-class liquid
• Mass spectral deconvolution was performed using the maximum entropy (MaxEnt) algorithm in MassLynx software (version 4.1, Waters).
• the ReACT labeling reactions were performed at room temperature for 10 min and quenched by desalting twice to remove free oxaziridine probes.
• the CuAAC reactions were performed on the labeled protein with aforementioned method using 200 ⁇ acid- cleavable biotin azide probe (200x stock in DMSO).(40, 41) Samples were precipitated and washed with cold methanol and dissolved in 250 ⁇ of 2% SDS/PBS. The solutions were diluted to 5 mL with 1% triton X-100/PBS. The solutions were then added with 2 mg of streptavidin-coated magnetic beads (Promega Corporation) overnight at 4 °C with agitation. The magnetic beads were washed with 1% triton X-100/PBS (5 mL), PBS (5 mL), 6 M urea (5 mL) and PBS (5 mL).
• the washed beads were then reduced with 5 mM TCEP at 65 °C for 15 min and alkylated with 10 mM iodoacetamide (IAA) at 37 °C for 30 min.
• On-beads trypsin (2 ⁇ g each sample) digestion were performed at 37 °C for 16 hour.
• the beads were then pelleted and washed with PBS (2 x 2mL), water (2 x 2mL).
• Modified peptides on the magnetic beads were cleaved using 1% formic acid/water (2 x 500 iL) at room temperature for 30 min and subsequently cleaved with 1% formic acid + 50% acetonitrile/water (2 x 500 iL) with agitation for 30 min.
• the eluents were combined and concentrated with a vacuum concentrator.
• Peptide samples were desalted by PierceTM CI 8 Spin Columns (Thermo Fisher Scientific) and kept at - 20 °C until analysis.
• Rate constants k for Ox2 was measured under pseudo first order conditions with a 5- to 40-fold excess of N-acetyl- L-methionine in PBS buffer by following the exponential growth in UV absorbance of the benzaldehyde at 250 nm over time under kinetics model of UV-vis (Agilent
• MS survey scan was obtained for the m z range of 300-1600; MS/MS spectra were acquired using a top 15 method, where the top 15 ions in the MS spectra were subjected to High Energy Collisional Dissociation (HCD).
• HCD High Energy Collisional Dissociation
• An isolation mass window of 1.6 m/z was used for the precursor ion selection, and normalized collision energy of 27% was used for fragmentation. Five second duration was used for the dynamic exclusion.
• Tandem identifications required at least -Log (Expect Scores) scores of greater than 1.2 with a mass accuracy of 5 ppm. Protein identifications were accepted if they contained at least 2 identified peptides. Using the parameters above, the Decoy False Discovery Rate (FDR) was calculated to be 1.1% on the protein level and 0.0% on the spectrum level.(42) Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. [0106] Colocalization experiments. Colocalization experiments were performed in live HEK293T cells with inducible cell surface GFP expression system. HEK293T cells were plated on poly- lysine coated chamber slides.
• Yeast genome editing S. cerevisiae strain BY4741 (201388; ATCC) was used as WT strain for genome editing. Knock out and mutation strains were generated by CRISPR-Cas9- mediated genome editing protocol as reported. (43) Briefly, for EN02 knock out strain, yeast competent cell (50 iL) was transformed with 1.0 ⁇ g pCAS plasmid and 2.0 ⁇ g linear repair DNA using Frozen-EZ Yeast Transformation IITM Kit (Zymo Research) according to manufacturer's protocol. The transformed cells were plated onto YPG plates with antibiotics. Cells were grown overnight at 37 °C and then another 48 h at 30 °C. The positive colonies ware confirmed by DNA sequencing. For EN01-M171L (EN02 null) strain, EN02 knock out strain (losing pCAS9 plasmid) was used as mother strain for subsequent enolase 1 mutation.
• GFP-Fabs production GFP-Fabs were produced using phage display methods and were constructed into a pSFV4 expression vector as previously described. (44). Methionine point mutations were incorporated by site-directed mutagenesis at heavy chain A114M, light chain V205M, and at the C-terminus of the light chain directly after the interchain disulfide. Recombinant Fabs were produced in C43 (DE3) Pro-i- cells as previously described. (44) Briefly, cultures were grown to OD -0.6 and induced with 0.2 mM IPTG at 30 °C overnight. Fabs were purified by protein A chromatography and buffer exchanged into PBS for subsequent storage and validation assays. For live cell application, endotoxin in recombinant Fabs solutions were removed by High Capacity Endotoxin Removal Spin Columns (Thermal Fisher Scientific).
• Her-Fabs production Light chain and heavy chain of Her-Fab were codon optimized(45) and constructed into a pComb3XSS expression vector as previously described. (46)
• Yeast enolase 1 production Yeast enolase 1 was amplified from yeast (S288C) genomic DNA and inserted into a pET28a expression vector with a 6x His tag at the N- terminus. Met 171 or 371 was converted to Leu using site-directed mutagenesis. The recombinant proteins were expressed in BL21(DE3)pLysS (Invitrogen) strain and purified according to previous papers. (47, 48) The sequence alignment of enolase family proteins was perform by the Clustal Omega program. (49)
• Oxl was synthesized with the same procedure used in making 0x2 using ethyl carbamate in place of 1-ethylurea in 20% yield. 3 ⁇ 4 NMR (400 MHz, CDCb) ⁇ 7.52
• Ox3 was synthesized with the same procedure used in making 0x2 using 2,2,2-trifluoro-l- phenylethan-l-one in place of benzaldehyde in 10% yield.
• Ox4 was synthesized with the same procedure used in making 0x2 using 4a as substrate in place of 1-ethylurea in 21% yield.
• CiiHiiN 2 02 + requires 203.0815.
• the amine 5a was synthesized according literature.(50) To a solution of amine 5a (1.9 g, 18.8 mmol) in aqueous HCl soulution (1.0 N, 19 mL) was added KOCN (7.6 g, 94 mmol) at r.t. After stirring overnight at 60 °C, the mixture was cooled to 0 °C. The mixture was extracted with ethyl acetate repeatedly until full extraction which was determined by TLC analysis. The organic layer was dried over Na2S0 4 , filtered and concentrated under vacuum to give a residue. To a solution of the residue in MeOH (50 mL) was added silica gel (10 g) at r.t.
• Ox5 was synthesized with the same procedure used in making 0x2 using 5b as substrate in place of 1-ethylurea in 33% yield.
• l H NMR 400 MHz, CDCh
• 5.00 s, 1H
• 3.67 - 3.57 m, 2H)
• Sulfimide S3 can be protonated under strong acidic condition to its salt form S4, which is stable in aqueous solution.
• Isotope-Targeted Glycoproteomics (IsoTaG): A Mass-Independent Platform for Intact N- and O- glycopeptide Discovery and Analysis. Nature Methods 12, 561-567, (2015).

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  • 2017-11-13 WO PCT/US2017/061412 patent/WO2018089951A1/en not_active Ceased
• 2019
  • 2019-05-02 US US16/402,113 patent/US11353463B2/en active Active

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