WO2024081302A1 - Modification covalente dirigée par un ligand de chaînes latérales d'acides aminés à l'aide d'électrophiles de mannich d'imine cyclique - Google Patents

Modification covalente dirigée par un ligand de chaînes latérales d'acides aminés à l'aide d'électrophiles de mannich d'imine cyclique Download PDF

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WO2024081302A1
WO2024081302A1 PCT/US2023/034916 US2023034916W WO2024081302A1 WO 2024081302 A1 WO2024081302 A1 WO 2024081302A1 US 2023034916 W US2023034916 W US 2023034916W WO 2024081302 A1 WO2024081302 A1 WO 2024081302A1
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protein
ligand
tyrosine
lysine
imine
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PCT/US2023/034916
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Casey John Krusemark
Emily C. DYKHUIZEN
Sijie WANG
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Purdue Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • TECHNICAL FIELD [0003] The present disclosure relates to methods of ligand-directed, covalent modification of tyrosines, tryptophans, and pyrrolated lysines in proteins using cyclic imine Mannich electrophiles.
  • BACKGROUND [0004] The number and diversity of covalent inhibitors has increased dramatically in recent years. The majority are targeted covalent inhibitors (TCIs), which are rationally designed to bind to a protein target and then form a covalent bond. A TCI brings a reactive moiety (typically an electrophile of low reactivity) directly adjacent to an amino acid side chain, inducing a high effective concentration.
  • TCIs targeted covalent inhibitors
  • PPIs protein-protein interfaces
  • Cysteines are rare amino acids and are also poorly represented in PPIs (Way et al. Curr. Opin. Chem. Biol.2000, 4(1), 40-46; Arkin et al. Chem. Biol.2014, 21(9), 1102-1114; Cheng et al. J. Hematol. Oncol.2020, 13(1), 1-14; Abbasov et al. Nat. Chem.2021, 13(11) 1081-1092). Development of additional chemical warheads for targeting additional residues will further expand the utility of TCIs (Gehringer et al. J. Med. Chem.2019, 62(12), 5673-5724; Mukherjee et al. Curr. Opin. Chem.
  • Tyrosines are attractive targets due to their functions in non-covalent interactions including hydrogen bonding, cation-pi or pi-pi interactions, and hydrophobic interactions (Koide et al. ACS Chem. Biol.2009, 4(5), 325-334; Hett et al. ACS Chem. Biol.2015, 10(4), 1094-1098; Gambini et al. J. Med. Chem.2019, 62(11), 5616-5627) . While tyrosine is an amino acid of low abundance (3% of protein amino acids) (Bartesaghi et al.
  • Aryl diazonium salts also have been used to conjugate tyrosine, but high reactivity and poor stability limit their use in TCIs (Schlick et al. J. Am. Chem. Soc.2005, 127(11), 3718-3723; Hooker et al., J. Am. Chem. Soc.2004, 126(12), 3718-3719; Sengupta et al. Or. Biomol. Chem.2019, 17(36), 8308-8329) .
  • a method of covalently modifying a tyrosine, tryptophan or pyrrolated lysine in a protein comprising a ligand binding site comprises reacting the side chain of the tyrosine, tryptophan or pyrrol-lysine with a cyclic imine Mannich electrophile appended to a ligand that binds the protein.
  • the cyclic imine can have a structure selected from: .
  • the cyclic imine can be stable in aqueous conditions at a temperature of about 37 °C (e.g., 37 °C) for at least about 12 hours (e.g., 12 hours).
  • the reacting can be carried out in vitro, e.g., in phosphate-buffered saline, pH 7, at 37°C.
  • the reacting can be carried out in a cell, such as a cell in culture.
  • the present disclosure provides for a protein comprising a ligand binding site, which comprises one or more tyrosine, tryptophan or pyrrolated lysine residues, the side chains of which have been selectively covalently modified with a cyclic imine.
  • the ligand binding site can further comprise one or more amino acid residues selected from the group consisting of lysine, histidine, serine, threonine, cysteine, glutamic acid, aspartic acid, an 69838-02 N-terminus of the protein, and a C-terminus of the protein, none of which are covalently modified irreversibly with the cyclic imine.
  • the present disclosure also provides for a protein comprising a ligand binding site, which comprises one or more tyrosine, tryptophan or pyrrolated lysine resides, the side chains of which have been selectively covalently modified by the method disclosed herein.
  • the present disclosure also provides for a cyclic imine Mannich electrophile appended to a ligand that binds to a target protein, wherein the modified ligand is suitable for reacting the side chain of the tyrosine, tryptophan or pyrrole-lysine of the target protein with the cyclic imine Mannich electophile appended ligand. It is further provided that the modified ligand is bound by a target protein active site. It is further provided that the modified ligand is bound by the target protein active site with measurable specificity.
  • the present disclosure also provides for a kit for covalently modifying a tyrosine, tryptophan, or pyrrolated lysine of a target protein, the kit comprising a cyclic imine Mannich electrophile appended to a ligand that binds the protein, suitable for reacting the side chain of the tyrosine, tryptophan or pyrrole-lysine of the target protein with the cyclic imine Mannich electophile appended ligand.
  • the present disclosure also provides for a kit for covalently modifying a tyrosine, tryptophan, or pyrrolated lysine of a target protein, the kit comprising a reagent comprising a cyclic imine Mannich electrophile suitable for being appended to a ligand that binds a target protein; a reagent suitable for reacting the side chain of the tyrosine, tryptophan or pyrrole-lysine of a target protein with a cyclic imine Mannich electophile appended ligand.
  • Fig.1 compares the provided method with the prior art.
  • Fig.2 shows the reaction of cyclic imine Mannich electrophiles (5 mM) with N-acetyl- 1-tyrosine methyl amide (100 mM) in phosphate-buffered saline (PBS), pH 7.0, at 37°C. The reaction was monitored using liquid chromatography/mass spectrometry (LC/MS).
  • Fig. 3 shows second-order rate constants for select cyclic imines with aromatic nucleophiles. Reactions were conducted under pseudo first order conditions with an excess of one reactant. The reduction in the concentration of the limiting reactant A was measured at various time points by HPLC analysis.
  • Fig. 4 shows representative HPLC analysis for second-order rate constants. Reaction 69838-02 of imine 1 (5 mM) with (A) N-Ac-Tyr-NHMe (100 mM) or (B) indole-3-butyrate (100 mM) at indicated time points. [0022] Fig. 5 shows second order rate constant determinations.
  • Fig.6 shows testing of additional amino acid functional groups for reactivity to imine 1.
  • Cyclic imine 1 was incubated with 100 mM compounds in PBS (pH 7) at 37°C for 24 hours and then analyzed by LC/MS.
  • A Compounds include ⁇ -mercaptoethanol, imidazole, isopropanol, n-butylamine, propionic acid, 3-methyl indole, and N-ethyl pyrrole (pyrrole-lysine).
  • Fig.7 shows the stability of the tyrosine Mannich product. After 24 hours of test reactions in PBS (pH 7) at 37°C, reaction mixture was further incubated with 1% TFA or 1% NaOH or PBS buffer for 96 hours.
  • Fig.8 shows the reversibility of the thiol-imine reaction. Cyclic imine 1 was reacted with 100 mM cysteine to pre-form the imine-cysteine conjugate, followed up with 1 M (10 eq) glutathione (GSH), another cysteine containing molecule, or 100 mM (1 eq) acetyl tyrosine methyl amide. Initial imine-cysteine product (imine-thiol product m/z calc.
  • Cyclic imine 1 (5 mM) was incubated with 100 mM acetyl tyrosine methyl amide and 5 mM GSH (in the presence of 5 mM TCEP). Reaction yield was quantified based on UV integrations of Mannich product at 4h, 9h, 17h, 24h, and 32h. Imine consumption rate was quantified based on the UV integration of Mannich product at 4h, 9h, 17h, 24h, and 32h. 69838-02 [0027] Fig.10 shows the reaction of imine 1 with tyrosine containing peptide angiotensin II. UV trace of reaction mixture of compound 1 (cyclic imine 1) with angiotensin II.
  • Angiotensin II (10 mM) was incubated with imine 1 (50 mM) for 16 hours prior to LC/MS analysis.
  • Di- cyclic imine-angiotensin II conjugate peak at 2.1 min calc.
  • Fig.12 shows validation of cyclic imine inhibitor P1b stability.
  • Cyclic imine inhibitor P1b was formed by treatment of P1bL with sodium periodate, and the crude sample in PBS was allowed to incubate for 24 hours. The sample was split, and one portion was further reduced with sodium cyanoborohydride.
  • LC/MS UV traces, low resolution mass spectrum.
  • LR-ESI-MS m/z calc. for P1bL [M+H] + .: 924.6, found: 924.9; m/z calc. for P1b [M+H] + : 875.5, found: 875.8; m/z calc.
  • FIG.13 shows the characterization of ligand directed protein labeling with iminolactone inhibitor P2.
  • A The fraction of labeled protein as determined by UV integration on reverse phase HPLC. CBX8 ChD and P2 were incubated at 10 ⁇ M each for 16 hours at 37°C. UV absorption measured at 215 nm.
  • B Mass spectra of CBX8 ChD treated overnight with inhibitor P2 (top, red) and control CBX8 ChD (bottom, green).
  • a method of covalently modifying a tyrosine, tryptophan, or pyrrolated lysine in a protein comprising a ligand binding site comprises reacting the side chain of the tyrosine, tryptophan or pyrrole-lysine with a cyclic imine Mannich electrophile appended to a ligand that binds the protein.
  • the cyclic imine can have a structure selected from:
  • the cyclic imine can be stable in aqueous conditions at a temperature of about 37 °C (e.g., 37 °C) for at least about 12 hours (e.g., 12 hours).
  • Alkyl generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms (e.g., C1-C15 alkyl).
  • Alkyl is intended to include independent recitations of a saturated “alkyl, " unless otherwise stated.
  • An alkyl can comprise one to thirteen carbon atoms (e.g., C1-C13 alkyl).
  • An alkyl can comprise one to eight carbon atoms (e.g., C1-C8 alkyl).
  • An alkyl can comprise one to five carbon atoms (e.g., C 1 -C 5 alkyl).
  • An alkyl can comprise one to four carbon atoms (e.g., C1-C4 alkyl).
  • An alkyl can comprise one to three carbon atoms (e.g., C1-C3 alkyl).
  • An alkyl can comprise one to two carbon atoms (e.g., C1-C2 alkyl).
  • An alkyl can comprise one carbon atom (e.g., C 1 alkyl).
  • An alkyl can comprise five to fifteen carbon atoms (e.g., C5-C15 alkyl).
  • An alkyl can comprise five to eight carbon atoms (e.g., C 5 -C 8 alkyl).
  • An alkyl can comprise two to five carbon atoms (e.g., C 2 -C 5 alkyl).
  • An alkyl can comprise three to five carbon atoms (e.g., C3-C5 alkyl).
  • the alkyl group can be selected from methyl, ethyl, 1-propyl (n-propyl), 1- methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl 69838-02 (iso-butyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl (n-pentyl).
  • the alkyl is attached to the rest of the molecule by a single bond.
  • Aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
  • the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) pi–electron system in accordance with the Hückel theory.
  • the ring systems from which aryl groups are derived include, but are not limited to, benzene, fluorene, indane, indene, tetralin and naphthalene.
  • the reacting is carried out under conditions that enable or promote the reaction of the side chain of the tyrosine, tryptophan or pyrrole-lysine with the cyclic imine Mannich electrophile appended to a ligand that binds to the protein.
  • the reacting can be carried out in vitro, e.g., in phosphate-buffered saline, pH 7, at 37°C.
  • the reacting can be carried out in a cell, such as a cell in culture.
  • the method can be used with any protein comprising a ligand binding site.
  • the ligand can be any ligand with which the protein interacts, whether under normal or abnormal conditions (e.g., diseased conditions).
  • the ligand can be one synthesized to interact with the protein, such as with the ligand binding site of the protein or another site which, upon binding, modulates (e.g., inhibits or promotes) the ability of the ligand binding site to bind a ligand.
  • the reaction of the cyclic imine Mannich electrophile with the side chain of a tyrosine, tryptophan or pyrrole-lysine in or near (e.g., adjacent) the ligand binding site results in a covalent modification, which, depending on the nature of the protein-ligand interaction and any downstream effect(s) of that interaction, can inhibit (i.e., down-regulate or turn “off”) or activate (i.e., up-regulate or turn “on”) the protein and, consequently, any downstream effect(s) of the change in activity of the protein.
  • the level of inhibition or activation is greater than that realized in the absence of covalent modification.
  • a protein comprising a ligand binding site, which comprises one or more tyrosine, tryptophan or pyrrolated lysine residues, the side chains of which have been selectively covalently modified with a cyclic imine.
  • the ligand binding site can further comprise one or more amino acid residues selected from the group consisting of lysine, histidine, serine, threonine, cysteine, glutamic acid, aspartic acid, an N-terminus of the protein, and a C-terminus of the protein, none of which are covalently modified irreversibly with the cyclic imine.
  • kits comprising reagents suitable for practicing the method as described above.
  • kits can comprise suitably prepared cyclic imine Mannich electophile reagents for linkage to protein target ligands.
  • kit could further comprise suitable supporting reagents, such as buffers, media and the like.
  • a kit is provided for where prepared ligand modified with cyclic imine Mannich electophile is a reagent that is prepared for use in the method as described above.
  • kits of the present disclosure can be prepared with complete sets of reagents providing for creation of target protein with one or more tyrosine, tryptophan or pyrrolated lysine residues, the side chains of which have been covalently modified with a cyclic imine.
  • Such reagents can be provided in bulk, in sets, individually or in other such suitable packaging.
  • This reaction rate is similar to the intrinsic rates of cysteine to acrylamides (1 x 10 -2 to 1 x 10 -4 M -1 s -1 ), which are commonly used in 69838-02 covalent drugs (Lonsdale et al. J. Chem. Inf. Model. 2017, 57(12), 3124-3137).
  • the ⁇ 10- fold increase in rate constant of 2 relative to 1 indicates the tunability of imine reactivity with adjacent electron withdrawing groups.
  • the tryptophan mimic 3-methyl indole, reacts with 1 to form multiple products, likely a mixture of N-substitution and C2/C3- Mannich- or Pictet Spangler-type additions (McFarland et al. J. Am. Chem. Soc.2008, 130(24), 7639-7644; Antos et al. J. Am. Chem. 69838-02 Soc.2004, 126(33), 10256-10257; Larda et al. Bioconjug. Chem.2015, 26(12), 2376-2383) (Fig. 6).
  • N-ethyl pyrrole as a mimic of pyrrolated lysine, a lesser-known post-translational modification (Miyashita et al. Sci. Rep.2014, 4, 5343; Chikazawa et al. J. Biol. Chem.2020, 295(22), 7697-7709) .
  • Pyrroles are particularly electron-rich aromatic groups that react rapidly in Mannich-type reactions.
  • Previous work with N- pyrrolyl alanine derivatives has shown fast and selective Pictet–Spengler reactions with aldehyde-containing biomolecules (Pomplun et al. Angew. Chemie Int.
  • Example 3 Stability and reversibility of cyclic imines with tyrosine and cysteine [0057] To validate the stability of cyclic imines as covalent warheads, imines 1-4 were evaluated by LC/MS after 96-hours incubation in PBS, 0.1M HCl, and 0.1M NaOH (Table 1). Compounds 1 and 4 were stable under all conditions. Both ester-containing compounds 2 and 3 hydrolyzed under basic conditions. Aromatic ester imine 3 was unstable in PBS, with a mixture of hydrolysis and Mannich reaction with the hydrolysis product 2-amino phenol.
  • the pre-formed thioaminal product of 1 with N-acetyl-cysteine methyl amide was treated with either glutathione (1 M) or Ac-Tyr-NHMe (100 mM). Both glutathione and Ac-Tyr-NHMe outcompeted the cysteine product after 24 hours (Fig.8).
  • glutathione and Ac-Tyr-NHMe outcompeted the cysteine product after 24 hours (Fig.8).
  • the cyclic imine-Tyr reaction (at 5 mM and 100 mM, respectively) was evaluated over time in the presence of cysteine (5 mM) (Fig.9). The presence of cysteine slightly slowed the reaction rate of tyrosine-imine compared to the control.
  • Example 4 Covalent labeling of cyclic imine with tyrosine-containing peptide (angiotensin II) and protein (CBX8 chromo-domain) [0061] To confirm reactivity and selectivity with a peptide context, imine 1 (50 mM) was incubated with the tyrosine-containing 8-mer peptide angiotensin II (DRVYIHPF) (10 mM) (Fig.10). The combined yield of mono-labeled and di-labeled angiotensin II was ⁇ 45% as quantified by LC/MS.
  • DUVYIHPF tyrosine-containing 8-mer peptide angiotensin II
  • CBX8 chromodomain CBX8 chromodomain
  • SW2_110A demonstrates high selectivity for CBX8 over CBX4, CBX6, and CBX7, it is only 6-fold selective over CBX2.
  • Amino acid sequence alignment and an X-ray crystal structure of a similar ligand (UNC3866) bound to CBX8 (Fig.11B) 69838-02 were used to identify a tyrosine (Y39) residue adjacent the diethyl lysine binding site that is a histidine in CBX2.
  • Example 6 Covalent labeling of cyclic imine containing ligands to CBX8 ChD [0069] To evaluate the reaction with CBX8 ChD, all cyclic imine ligands were tested with purified CBX8 ChD. To saturate the binding of inhibitor to protein, 10 ⁇ M of the covalent inhibitors were incubated with 10 ⁇ M CBX8 ChD ( ⁇ 10-fold over the K d ) in PBS buffer at 37 °C for 12 hours.
  • Example 7 [0071] Time-dependent fluorescence displacement assay [0072] A fluorescence polarization (FP) displacement assay was used to evaluate the covalent binding of cyclic imine warhead containing inhibitors. Without additional incubation time, the non-covalent inhibitor SW2_110A and optimal covalent inhibitors P1b ( iminolactam) and P2 (iminolactone) had comparable IC 50 values, which suggests that the structural modification of the warhead does not significantly change the affinity.
  • FP fluorescence polarization
  • CBX8 was robustly enriched using the biotinylated, covalent inhibitor P2-B.
  • P2-B protein significantly enriched with P2-B (log 2 (P2- B/DMSO) > 3, P ⁇ 0.001), only 69838-02 CBX8 enrichment was significantly reduced with competing non-reactive ligand SW2_110A, indicating ligand-dependent, covalent labeling of endogenous CBX8 from lysates. Results were similar using iminolactam inhibitor P1bP.
  • Example 10 [0082] Cellular activity of covalent inhibitors in MLL-AF9 transformed leukemia cell line [0083] Imine inhibitors were assessed for improved cellular activity using a disease-relevant cell model.
  • inhibitor P1b was the most effective at reducing HOXA9 gene expression where 10 ⁇ M P1b treatment reduced gene expression to a similar extent as 100 ⁇ M SW2_110A (Fig.15A).
  • the two optimal inhibitors P1bP and P2 were subsequently tested for dose-dependency in this cellular assay. Dose curves indicate that these covalent inhibitors have 30 to 100-fold improvement of cellular activity compared to the non-covalent inhibitor (Fig.15B).
  • references to “the method” includes one or more methods and/or steps of the type, which are described herein and/or which will become apparent to those ordinarily skilled in the art upon reading the disclosure.

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Abstract

L'invention concerne un procédé de modification covalente de tyrosines, de tryptophane et de lysines pyrrolatées dans une protéine comprenant la réaction des chaînes latérales des acides aminés avec un électrophile de Mannich d'imine cyclique ajouté à un ligand qui se lie à la protéine ; et une protéine comprenant un site de liaison de ligand, qui comprend un ou plusieurs résidus tyrosine, tryptophane ou lysine pyrrolatée, dont les chaînes latérales ont été sélectivement modifiées de manière covalente avec une imine cyclique.
PCT/US2023/034916 2022-10-14 2023-10-11 Modification covalente dirigée par un ligand de chaînes latérales d'acides aminés à l'aide d'électrophiles de mannich d'imine cyclique WO2024081302A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US20170218353A1 (en) * 2009-12-30 2017-08-03 Celgene Car Llc Ligand-Directed Covalent Modification of Protein
US20210300972A1 (en) * 2018-10-31 2021-09-30 Ajinomoto Co., Inc. Compound having affinity substance to antibody, cleavable portion, and reactive group, or salt thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170218353A1 (en) * 2009-12-30 2017-08-03 Celgene Car Llc Ligand-Directed Covalent Modification of Protein
US20210300972A1 (en) * 2018-10-31 2021-09-30 Ajinomoto Co., Inc. Compound having affinity substance to antibody, cleavable portion, and reactive group, or salt thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Substance record 4 December 2011 (2011-12-04), ANONYMOUS: "CID 18177643 (5,6-Dihydropyrazine-2(1h)-one)", XP093163808, Database accession no. SID 129202717 *
WAN G: "Development of Chemical Probes to CBX Chromodomain Using DNA- Encoded Libraries and Covalent Conjugation with Mannich Electrophiles", PH.D., THESIS, August 2022 (2022-08-01), pages 110 - 131, Retrieved from the Internet <URL:https://hammer.purdue.edu/articles/thesis/DEVELOPMENT_OF_CHEMICAL_PROBES_TO_CBX_CHROMODOMAIN_USING_DNA-ENCODED_LIBRARIES_AND_COVALENT_CONJUGATION_WITH_MANNICH_ELECTROPHILER/20373330> [retrieved on 20231218] *

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