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US-20260124313-A1 - SITE-SPECIFIC COVALENT LIGATION OF HUMAN SERUM ALBUMIN

US20260124313A1US 20260124313 A1US20260124313 A1US 20260124313A1US-20260124313-A1

Abstract

Provided herein are albumin selective peptides for covalent ligation to human serum albumin, and conjugates thereof.

Inventors

  • Kit S. Lam
  • XINGJIAN YU
  • Ruiwu Liu

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260507
Application Date
20231006

Claims (20)

  1. 1 . A compound of Formula I: or a pharmaceutically acceptable salt thereof, wherein R 1 is a targeting moiety; Peptide comprises 3 to 10 amino acids; L 1 is a linker; R 2 is a drug, imaging agent, or affinity ligand; and R 3 is hydrogen or C 1-6 alkyl.
  2. 2 . The compound of claim 1 , wherein the targeting moiety is specific to a binding site of human serum albumin (HSA).
  3. 3 . The compound of claim 2 , wherein the HSA binding site is Sudlow I or Sudlow II.
  4. 4 . The compound of any one of claims 1 to 3 , wherein the targeting moiety is a fatty acid, ibuprofen, or iophenoxic acid.
  5. 5 . The compound of any one of claims 1 to 3 , wherein at least one amino acid of the Peptide is lysine.
  6. 6 . The compound of any one of claims 1 to 5 , having Formula II: wherein R 1 is a fatty acid; X 4 is absent, Aad, D, d, E, e, A, a, V, v, L, I, i, L, l, M, m, F, f, Y, y, W, or w; X 3 is Aad, D, d, E, or e; K is lysine; X 2 is S, s, T, t, N, n, Q, q, Aad, D, d, E, e; X 1 is Aad, D, d, E, e, A, a, V, v, L, I, i, L, l, M, m, F, f, Y, y, W, w, S, s, T, t, N, n, Q, or q; L 1 is a linker; R 2 is the drug, imaging agent, or affinity ligand; and R 3 is hydrogen or C 1-6 alkyl.
  7. 7 . The compound of claim 6 , wherein the fatty acid is a saturated fatty acid.
  8. 8 . The compound of claim 6 or 7 , wherein the fatty acid is caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid.
  9. 9 . The compound of any one of claims 6 to 8 , wherein the fatty acid is myristic acid.
  10. 10 . The compound of any one of claims 6 to 9 , wherein at least two of X 4 , X 3 , X 2 and X 1 are each independently Aad, D, d, E, or e.
  11. 11 . The compound of any one of claims 6 to 10 , wherein X 4 is absent, e or a; X 3 is Aad, D, or e; X 2 is s, Aad, D, or e; and X 1 is Aad, Y, e, or s, wherein at least two of X 4 , X 3 , X 2 and X 1 are each independently Aad, D, or e.
  12. 12 . The compound of any one of claims 1 to 11 , wherein R 3 is hydrogen or methyl.
  13. 13 . The compound of any one of claims 1 to 12 , wherein R 3 is hydrogen.
  14. 14 . The compound of any one of claims 6 to 13 , having Formula IIa: wherein R 1 is myristic acid; X 4 is absent, e or a; X 3 is Aad, D, or e; K is lysine; X 2 is s, Aad, D, or e; X 1 is Aad, Y, e, or s; L 1 is a linker; and R 2 is the drug, imaging agent, or affinity ligand, wherein at least two of X 4 , X 3 , X 2 and X 1 are each independently Aad, D, or e.
  15. 15 . The composition of any one of claims 1 to 14 , wherein the compound is: wherein L 1 is the linker and R 2 is the drug, imaging agent, or affinity ligand.
  16. 16 . The compound of any one of claims 6 to 13 , having Formula III: wherein R 1 is ibuprofen or iophenoxic acid; X 4 is absent, Acpc, Aib or N; K is lysine; X 3 is W, Nle, R, or P; X 2 is HoCit, R, I, HyP, or Acpc; X 1 is R, HoPhe, Nle, T, or N; L 1 is a linker; and R 2 is the drug, imaging agent, or affinity ligand, wherein at least one of X 4 , X 3 , X 2 and X 1 is Acpc, W, Aib, Nle, HoPhe, or I.
  17. 17 . The compound of claim 16 , having Formula III: wherein R 1 is ibuprofen; X 4 is absent, Acpc, Aib or N; K is lysine; X 3 is W, Nle, or R; X 2 is HoCit, R, or I; X 1 is R, HoPhe, or Nle; L 1 is a linker; and R 2 is a drug, imaging agent, or affinity ligand, wherein at least two of X 4 , X 3 , X 2 and X 1 are each independently Acpc, W, Aib, Nle, HoPhe, or I.
  18. 18 . The composition of claim 16 or 17 , wherein the compound is: wherein L 1 is the linker and R 2 is the drug, imaging agent, or affinity ligand.
  19. 19 . The compound of claim 16 , having Formula III: wherein R 1 is iophenoxic acid; X 4 is Aib; K is lysine; X 3 is R, or P; X 2 is HyP, or Acpc; X 1 is T, or N; L 1 is a linker; and R 2 is a drug, imaging agent, or affinity ligand, wherein at least one of X 4 , X 3 , X 2 and X 1 is Acpc, or Aib.
  20. 20 . The composition of claim 16 or 19 , wherein the compound is: wherein L 1 is the linker and R 2 is the drug, imaging agent, or affinity ligand.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/378,605, filed Oct. 6, 2022, which is incorporated herein in its entirety for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant Nos. 1R21AI132458-01 awarded by NIH/NIAID and R01CA247685 awarded by NIH/NCI. The Government has certain rights in this invention. BACKGROUND Human serum albumin (HSA) is the most abundant protein in human blood plasma. There have been many successful precedences leveraging the advantages of HSA as a platform for different diagnostic and therapeutic applications. HSA has been successfully used clinically as a non-covalent carrier for insulin (e.g. Levemir), GLP-1 (e.g. Liraglutide), and paclitaxel (e.g. Abraxane). Efforts have been made to use HSA as a covalent carrier for drug delivery. However, none has been approved for clinical use so far. The development of post-translational chemical modifications that can derivatize native HSA site-specifically under mild reactions will allow researchers to engineer HSA-drug conjugates or supramolecular HSA-based nanostructures, with desirable pharmacokinetic (PK)/pharmacodynamic (PD) properties and protein-adduct ratio, for various biomedical applications. The HSA's only free cysteine (Cys34) makes maleimide chemistry a viable approach to site-specifically modify HSA. One of the concerns for the maleimide-based conjugation strategy, however, is that the resulting thiosuccinimide linkage is unstable under reducing conditions through the retro retro-Michael reaction, or thiol exchange, which poses risks in the performance and safety of the HSA conjugates due to possible unexpected payload release. Furthermore, for targeted drug delivery using HSA as the carrier, it would be advantageous to have additional site-specific ligation strategies that are orthogonal to Cys34, such that targeting ligands and one or more payloads can be reliably and site-specifically conjugated to HSA to form a homogenous conjugate. The ε-amine on the lysine side chain is another popular site for protein conjugation. The cationic nature of lysine residues at physiological pH makes their distribution relatively on the protein surface and more accessible to conjugation reagents. The conventional lysine conjugation strategy is through reaction with electrophiles, such as N-hydroxysuccinimide ester (NHS-ester), isothiocyanate, or activated aromatic esters. Site-specific conjugation to lysine is more challenging as lysines are abundant in the proteome. For example, for HSA there are 48 lysines in total. These highly reactive electrophiles often fail to differentiate particular lysines, and they tend to react with lysines randomly. Using these non-specific bioconjugation techniques to prepare protein conjugates may compromise the properties of proteins by accidentally labeling physiologically important amino acids. For example, domain IIIB and domain I of HSA are known to be essential for their binding to cell surface FcRn, which is responsible for the recycling of HSA. Modification of surface lysines on these two HSA domains will likely lead to a decrease in circulation half-life of HSA. In the case of antibody-drug conjugates (ADC), non-specific bioconjugation techniques involving lysines could bring unexpected cytotoxicity to the protein conjugates, while on the contrary, homogenous ADCs prepared by site-specific approaches have demonstrated improved therapeutic index. Nevertheless, there have been several studies on performing site-specific conjugation on lysine residues, and site-specific ligations to HSA utilizing Aza-Michael addition between the ε-amine of lysine residues and Michael acceptors, such as α, β-unsaturated sulfonamides and sulfonyl acrylate have also been reported. The chemoselectivity and site-specificity of these strategies originate from kinetic control or delicately tuned substrates such that lysine with higher nucleophilicity and better accessibility can react preferentially. These generic bioconjugation methods, however, are reactive to a variety of proteins. For in vivo applications, it is demanding to develop HSA-exclusive methods to avoid off-target ligation. Surprisingly, the present invention meets this and other needs. BRIEF SUMMARY OF THE INVENTION In one embodiment, the present invention provides a compound of Formula I: wherein R1 is a targeting moiety;Peptide comprises 3 to 10 amino acids;L1 is a linker;R2 is a drug, imaging agent, or affinity ligand; andR3 is hydrogen or C1-6 alkyl. In another embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention, and a pharmaceutically acceptable excipient. In another embodiment, the present invention provides a conjugate of Formula IV: wherein R1 is a targeting moiety;Peptide comprises 3 to 10 amino acids;L1 is a l