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Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

ABSTRACT: The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long serum half-life and can be used to extend the serum half-lives of therapeutic proteins and peptides. We used glucagon-like-peptide 1 (GLP-1) as a model peptide in the present study. A "clickable" non-natural amino acid-p-azido-l-phenylalanine (AzF)-was incorporated into three specific sites (V16, Y19, and F28) of a GLP-1 variant, followed by conjugation with HSA through strain-promoted azide-alkyne cycloaddition. All three HSA-conjugated GLP-1 variants (GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA) exhibited comparable serum half-lives in vivo. However, the three GLP1_HSA variants had different in vitro biological activities and in vivo glucose-lowering effects, demonstrating the importance of site-specific HSA conjugation. The platform described herein could be used to develop other therapeutic peptides with extended serum half-lives.

SUBMITTER: Bak M

PROVIDER: S-EPMC7238188 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

Bak Mijeong M Park Junyong J Min Kiyoon K Cho Jinhwan J Seong Jihyoun J Hahn Young S YS Tae Giyoong G Kwon Inchan I

Pharmaceutics 20200416 4

The number of therapeutic peptides for human treatment is growing rapidly. However, their development faces two major issues: the poor yield of large peptides from conventional solid-phase synthesis, and the intrinsically short serum half-life of peptides. To address these issues, we investigated a platform for the production of a recombinant therapeutic peptide with an extended serum half-life involving the site-specific conjugation of human serum albumin (HSA). HSA has an exceptionally long se ...[more]

PMID: 32316169

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Project description:The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host-cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT-gp120 complex as a blueprint to design a covalently conjugated PT-gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21-E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT-gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT-gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT-gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT-gp120 conjugate to the noncovalent PT-gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT-gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced "off" state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.

Dataset Information

Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

Publications

Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide.

Similar Datasets

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