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JP-2026076184-A - Polyfunctional chimeric molecules

JP2026076184AJP 2026076184 AJP2026076184 AJP 2026076184AJP-2026076184-A

Abstract

[Problem] To provide a polyfunctional chemical conjugation molecule that can be found to be useful as a modifier for a target substrate. [Solution] A polyfunctional chemical conjugation molecule is provided, comprising a localization portion, a chemical linker portion, an activator portion, a first orientation adapter that interconnects the chemical linker portion to the activator portion at one end, and optionally a second orientation adapter that interconnects the chemical linker molecule to the localization portion at a different end. The molecule provides a use for post-translational modification of a polymer that is not a natural substrate of the activator portion. Diseases or disorders may be treated or prevented by this molecule. [Selection Diagram] None

Inventors

  • チョーダリー アミット
  • レイ ソフィア
  • ショーバ ヴェロニカ
  • コッコンダ プラヴィーン
  • ムンカナッタ ゴダージュ ダヌシカ
  • シリワルデナ サチーニ

Assignees

  • ザ・ブロード・インスティテュート・インコーポレイテッド
  • ザ ブリガム アンド ウィメンズ ホスピタル インコーポレイテッド

Dates

Publication Date
20260511
Application Date
20251226
Priority Date
20200108

Claims (20)

  1. A polyfunctional chemical conjugation molecule comprising a localization portion, a chemical linker portion, an activator portion, a first orientation adapter connecting the chemical linker portion to the activator portion at one end, and optionally a second orientation adapter connecting the chemical linker molecule to the localization portion at a different end.
  2. Formula I-A Loc-L-(V 1 -Act) n (IA) (wherein Loc includes the localization portion, L is the chemical linker portion, V1 is the first orientation adapter, and Act is the activator portion, and n is at least 1); or formula I-B Loc-V 2 -L-(V 1 -Act) n (I-B) (In the formula, Loc includes the localization portion, L is the chemical linker portion, V1 is the first orientation adapter, V2 is the second orientation adapter, and Act is the activator portion.) The molecule according to claim 1, represented by [the given expression].
  3. The first and second orientation adapters are independently selected from Table 2, the molecule according to claim 1 or 2.
  4. The molecule according to claim 1, wherein the activating agent portion binds to an enzyme that modifies a target substrate associated with the localization portion, and activates it.
  5. The molecule according to claim 4, wherein, if the target substrate is not a native substrate of the enzyme, or if the activation of the enzyme by the activator molecule is not activated by binding to the activator moiety, the enzyme modifies the target substrate at one or more new modification sites that would otherwise remain unmodified by the enzyme.
  6. Linker, (In the formula, n is between 1 and 50.) A molecule selected from the following, according to claim 1.
  7. The molecule according to claim 1, wherein the linker is a PEG molecule, an alkyl, a heterocycloalkyl, a cycloalkyl, an aryl, an alkylene, an alkenyl, a heteroaryl, an amide, an amine, a thiol, or a derivative thereof.
  8. The molecule according to claim 1, wherein the linker is a polyfunctional linker.
  9. The molecule according to claim 8, wherein the linker is a polyfunctional PEG linker.
  10. The molecule according to claim 2, wherein n is 2 to 5.
  11. The activator portion of the molecule according to claim 1 has the ability to locate and activate an enzyme.
  12. The molecule according to claim 11, wherein the enzyme is a kinase, phosphatase, transferase, or ligase.
  13. The molecule according to claim 12, wherein the kinase is a serine/threonine kinase, a tyrosine kinase, or a bispecific protein kinase that phosphorylates the protein serine/threonine and the protein tyrosine.
  14. The molecule according to claim 13, wherein the kinase is AMP-activated protein kinase (AMPK), glucokinase (GK), or AGC kinase.
  15. The molecule according to claim 1, wherein the activating agent portion binds to protein kinase C (PKC) and activates it.
  16. The molecule according to claim 15, wherein the activating agent portion binds to a PKC isoform selected from PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-ε, PKC-δ, PKC-η, or PKC-ζ, and activates it.
  17. The activating agent portion is the molecule according to claim 15, selected from Table 2.
  18. The molecule according to claim 1, wherein the localization portion targets nucleic acids, polypeptides, or polysaccharides.
  19. The molecule according to claim 1, wherein the localization portion is a target polypeptide binding portion.
  20. The molecule according to claim 19, wherein the target polypeptide binding portion binds to a target polypeptide comprising a bromodomain and an extra-terminal motif (BET).

Description

Cross-reference of Related Applications This application claims the benefits of U.S. Provisional Patent Application No. 62/958,696 filed on 8 January 2020, U.S. Provisional Patent Application No. 63/057,879 filed on 28 July 2020, and U.S. Provisional Patent Application No. 63/069,655 filed on 24 August 2020. All the contents of the above-mentioned applications are incorporated herein by reference in their entirety. Reference to the Electronic Sequence Listing The contents of the electronic sequence listing ("BROD-4850WP_ST25.txt"; size 14,782 bytes, created on January 5, 2021) are incorporated herein by reference in their entirety. The subject matter disclosed herein generally relates to polyfunctional chemical conjugation molecules used to induce modifications in target substrates. Protein kinases regulate essential cellular functions such as the cell cycle, metabolism, differentiation, proliferation, and apoptosis. Kinase dysfunction is associated with various human diseases, including cancer, inflammatory conditions, autoimmune disorders, and heart disease. Figure 1A illustrates the formation of PHICS-induced ternary complexes between AMPK and BRD4 (PHICS 1.2) or between PKC and BRD4 (PHICS 2.3), and Figure 1C illustrates the schematic explanation of how proximity-induced phosphorylation is promoted by this chimeric molecule. Biochemical characterization of PHICS1.2-induced BRD4 phosphorylation by AMPK. (Figure 2A) Ternary complex formation of BRD4, PHICS, and AMPK observed by AlphaScreen (normalized with DMSO). (R)-PHICS1.2 is an inactive analog with low affinity for BRD4. (Figure 2B) ADP-Glo® assay for AMPK-catalyzed phosphorylation of BRD4 by PHICS1.2 compared with (R)-PHICS1.2. (Figure 2C) Western blot analysis of BRD4 phosphorylation by PHICS1.2 using a phospho-AMPK substrate motif antibody. (Figure 2D) Bell-type dependence of BRD4 phosphorylation as a function of PHICS1.2 concentration, analyzed by Western blot. (Figure 2E) ADP-Glo® assay for AMPK-mediated phosphorylation of various peptide sequences from BRD4 or peptides derived from the AMPK substrate ACC (SAMS peptide). (Figure 2F) Effect of AMPK isoforms on PHICS1.2-mediated BRD4 phosphorylation. (Figure 2G) AlphaScreen for ternary complex formation between AMPK, PHICS1.2, and various BRD proteins. (Figure 2H) Detection of PHICS1.2-catalyzed phosphorylation of various BRD proteins by Western blotting. The loading level of BRD proteins was determined by Kouma Siegel. Biochemical characterization of PKC-induced PHICS2.3-induced BRD4 phosphorylation. (Figure 3A) Formation of BRD4, PHICS, and PKC ternary complex observed by AlphaScreen normalized with DMSO control. (Figure 3B) ADP-Glo assay for PKC-catalyzed phosphorylation of BRD4 by PHICS2.3 compared with (R)-PHICS2.3. (Figure 3C) Detection of BRD4 phosphorylation by phospho-PKC substrate motif antibody in Western blot. (Figure 3D) Various BRD4 phosphorylation levels observed among PKC isoforms in the presence of PHICS2.3. (Figure 3E) Western blot analysis of PHICS2.3-mediated phosphorylation of various BRD proteins. (Figure 4A) Three-dimensional cocrystal structure of the activator PF-06409577 bound to AMPK, along with major interactions K29, K31, and D88 in the two-dimensional ligand map. (Figure 4B) Docking of the benzolactam activator and PKC, along with major interactions T242, L251, and G253 in the two-dimensional ligand map. Solvent-exposed sites where linkers are bound are highlighted in blue. A click chemistry platform for synthesizing PHICS1 with various linkers. Biochemical validation of AMPK activators, PHICS1 intermediates, and PHICS1.2 using an ADP Glo assay with SAMS peptide as a substrate. Biochemical validation of PHICS1 with various linkers to identify the optimal molecule for further research. (Figure 7A) Structures of PHICS1 analogs with various linker lengths. (Figure 7B) Schematic diagram of the Alpha screen assay for BRD4-PHICS-AMPK ternary complex formation. (Figure 7C) Alpha screen assay for PHICS1 with various linkers normalized with DMSO. (Figure 7D) Western blot analysis of AMPK-catalyzed BRD4 phosphorylation by PHICS1 analogs at various concentrations. Verification of inactive analogues. (Figure 8A) Structures of PHICS1.2 and its inactive analogue (R)-PHICS1.2. (Figure 8B) ADP-Glo with SAMSTide peptide as a substrate for comparison of AMPK activation by PHICS1 molecule. (Figure 8C) Formation of a ternary complex of AMPK and BRD4 by induction of PHICS1.2, observed by pull-down assay. (Figure 8D) Western blot analysis of His-tagged BRD4 (49-460) phosphorylation by AMPK in the presence of PHICS1.2. (Figure 8E) Effect of AMPK concentration on PHICS1.2 or (R)-PHICS1.2-mediated BRD4 phosphorylation, observed by Western blot. Mass spectrometry identification of BRD4 phosphorylation by AMPK in the presence of PHICS1.2. (Figure 9A) Spectra of T169, (Figure 9B) T186, (Figure 9C) T221, (Figure 9D) S324 and (Figure 9E) S325 phosphorylated peptides. Fragmentation patterns are show