Site-specific, kinetically inert conjugation of labels and/or carriers to target molecules such as his-tagged proteins via metal complex reagents

Patent metadata

Field	Value
Publication number	US-2022324893-A1
Application number	US-202017620087-A
Country	US
Kind code	A1
Filing date	Jun 18, 2020
Priority date	Jun 18, 2019
Publication date	Oct 13, 2022
Grant date	—

Abstract

The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) nitrate as a metal cation ligand and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention.

First claim

1 . A complex comprising:
a) a metal cation; b) nitrate as a metal cation ligand; and c) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier.

2 . The complex of claim 1 , wherein the chelating ligand of the metal cation chelating domain is a polydentate ligand that comprises one or more carboxylic acid groups and/or one or more amine groups and/or one or more aromatic amines and/or phosphates.

3 . The complex of claim 1 , wherein the chelating ligand of the metal cation chelating domain of c) is selected from:
nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptides such as peptides with the consensus sequence (GHHPH) n G; with n=1 to 3; see SEQ ID NOs: 1 to 3) or cadystin, triazacyclononane (TACN), diethylenetriamine-pentaacetate (DTPA), phytochelatin, carboxymethylaspartate (CMA), phosphonates, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionicdiacetic acid (NPDA), nitriloisopropionicdiacetic acid (NIPDA), N-(hydroxylethyl)ethylenediaminetriacetic acid (HEDTA), 1,4,7,10-tetraazacyclodo-decane-N,N′,N″,N′″-tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclonane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4.7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid (TETA), ethylenedicysteine, ethylenediaminetetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononane phosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyridimidine and derivatives thereof.

4 . The complex of claim 1 , wherein the chelating ligand of the metal cation chelating domain of c) is selected from: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), chelating peptides with the consensus sequence (GHHPH) n G, diethylenetriamine-pentaacetate (DTPA), nitrilopropionicdiacetic acid (NPDA), nitriloisopropionicdiacetic acid (NIPDA), ethylenediamine-tetraacetic acid (EDTA), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartate (CMA) and derivatives thereof.

5 . The complex of claim 1 , wherein the chelating ligand of the metal cation chelating domain of c) comprises or is selected from NTA, IDA and derivatives thereof.

6 . The complex of any one of claims 1 to 4 , wherein the metal cation is a transition metal cation.

7 . The complex of any one of claims 1 to 5 , wherein the metal cation of the complex is a di-, tri- or tetravalent metal cation.

8 . The complex of any one of claims 1 to 6 , wherein the metal cation is a metal cation having a water ligand exchange rate of 10 −1 s −1 or lower, preferably 10 −2 s −1 or lower.

9 . The complex of any one of claims 1 to 7 , wherein the metal cation is selected from the group consisting of: Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Pt 2+ , Pt 4+ , Ru 2+ , Ru 3+ , La 3+ , Eu 3+ , Os 2+ , Pd 4+ , Mo 3+ , Fe 3+ , Ru 3+ , Gd 3+ , Tc 3+ , Re 3+ , Sm 3+ , Tb 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Pm 3+ , Dy 3+ , Ho 3+ , Er 3+ , Tm 3+ , Yb 3++ , V 2+ , Mn 4+ , Fe 2+ and Lu 3+ .

10 . The complex of any one of claims 1 to 7 , wherein the metal cation is selected from the group consisting of: Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Ir 4+ , Pt 2+ , Pt 4+ , Pd 4+ , Mo 3+ , Fe 3+ , Gd 3+ , Tb 3+ , Eu 3+ , Ru 2+ , La 3+ , Ru 3+ , Re 3+ , Re 4+ , V 2+ , Mn 4+ , Fe 2+ and Os 2+ .

11 . The complex of any one of claims 1 to 7 , wherein the metal cation is Co 3+ or Pt 4+ .

12 . The complex of any one of claims 1 to 11 , wherein the complex comprises a [Pt(IV)(NTA)NO 3 ] − complex, wherein the label and/or carrier is attached to NTA.

13 . The complex of any one of claims 1 to 12 , wherein the label comprises a fluorophore, a diagnostic, a targeting moiety, a therapeutic agent, a PEG molecule, a lipid, biotin and/or its derivatives, proteins, peptides, a toxin and/or a reactive group selected from a thiol, azide, alkyne, nitrone, tetrazine and tetrazole.

14 . The complex of any one of claims 1 to 12 , wherein the label comprises or is a fluorophore.

15 . The complex of any one of claims 1 to 12 , wherein the label comprises or is biotin or derivatives thereof.

16 . The complex of any one of claims 1 to 12 , wherein the carrier is a polymer, a hydrogel, a microparticle, a nanoparticle, a sphere (including nano- and microsphere), a bead, a quantum dot, a prosthetic or a solid surface.

17 . The complex of any one of claims 1 to 16 , wherein the metal cation chelating domain comprises a linker between the chelating ligand and the label and/or carrier.

18 . A composition comprising a complex as defined in any one of claims 1 to 17 .

19 . Use of a complex of any one of claims 1 to 17 or a composition of item 18 for the labeling of a target molecule, wherein the target molecule comprises a protein, peptide or nucleic acid, preferably a protein or DNA that can exchange the metal cation ligand in the complex and even more preferably wherein said target molecule comprising at least 4 histidine residues or histidine-like residues in a sequence [H n S m ] k , wherein H is a histidine residue or a histidine-like residue, wherein S is a spacer amino acid residue, wherein n is in each case independently 1 to 4, wherein m is in each case independently 0 to 6, and wherein k is 2 to 6.

20 . Use of a complex of any one of claims 1 to 17 or a composition of claim 18 for the labeling of a target molecule, wherein said target molecule contains a histidine-rich region comprising at least two histidine residues, wherein said histidine-rich region is formed by a three-dimensional folding of the target molecule that brings said at least two histidine residues in spatial proximity, wherein the at least two histidine residues have a distance of 0 to 5 angstroms and are not consecutive in the amino acid sequence.

21 . The use of claim 20 , wherein the histidine-rich region is the Fc region of an antibody.

22 . Use of the complex of any one of claims 1 to 17 or a composition of item 18 for attaching a label and/or carrier to an antibody, a domain thereof (e.g. the Fc region) or fragments thereof.

23 . The use of claim 22 , wherein the label is a toxin.

24 . Use of a complex of any one of claims 1 to 17 or a composition of claim 18 for the labeling of a target molecule, said target molecule contains a region enriched in histidine-like residues, which develops during three-dimensional folding of the target molecule when histidine-like residues come in spatial proximity, wherein the at least two histidine-like residues have a distance of 0 to 5 angstroms and are not consecutive in the amino acid sequence.

25 . The use of claim 19 , 20 or 24 , wherein the target molecule is a pharmaceutical, a diagnostic, a research agent, a cosmetic and/or a protein for environment treatments (e.g. water treatment).

26 . The use of claims 19 , 20 or 24 , wherein the target molecule comprises or is a peptide or protein.

27 . The use of claim 19 , 20 , 24 , 25 or 26 , wherein the target molecule comprises or is an enzyme, a targeting protein such as an antibody, a cytokine, a transpo

Assignees

• Max Planck Gesellschaft
• Univ Heidelberg

Inventors

• Benk Amelie S
• Benk Lucia T
• Wegner Seraphine
• Comba Peter
• Spatz Joachim P

Classifications

• 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 (peptidic linkers A61K47/65) · CPC title

• G01N33/585

  with a particulate label, e.g. coloured latex · CPC title

• A61K47/547 Primary

  Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent · CPC title

• C07K1/13

  Labelling of peptides · CPC title

• C07F15/065 Primary

  without a metal-carbon linkage · CPC title