A biomimetic approach for enhancing the in vivo half-life of peptides

Journal name:
Nature Chemical Biology
Volume:
11,
Pages:
793–798
Year published:
DOI:
doi:10.1038/nchembio.1907
Received
Accepted
Published online

Abstract

The tremendous therapeutic potential of peptides has not yet been realized, mainly owing to their short in vivo half-life. Although conjugation to macromolecules has been a mainstay approach for enhancing protein half-life, the steric hindrance of macromolecules often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Here we report a new strategy for enhancing the in vivo half-life of peptides without compromising their potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Although there are a few molecules that bind albumin reversibly, we are unaware of designed small molecules that reversibly bind other serum proteins and are used for half-life extension in vivo. We show here that our strategy was effective in enhancing the half-life of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy.

At a glance

Figures

  1. Crystal structure of hTTR bound to AG10, and effect of binding to TTR on the half-life of AG10.
    Figure 1: Crystal structure of hTTR bound to AG10, and effect of binding to TTR on the half-life of AG10.

    (a) Crystal structure of hTTR bound to AG10, with monomers colored individually and a box showing close-up view of AG10 bound in one of the two hTTR T4 pockets (PDB ID: 4HIQ)22. (b) Percentage of AG10 (5 μM) remaining after 2 h incubation with human liver microsomes (HLM) in the absence and presence of hTTR (5 μM) or HSA (5 μM). Error bars represent the mean ± s.e.m. of three replicates. (c) Plasma concentration of AG10 after administering increasing doses of AG10 (single intravenous bolus of indicated concentrations) to three groups of rats (n = 3 per group). Error bars represent the mean ± s.e.m. of three biological replicates.

  2. TLHE1 and its peptide conjugates bind selectively to hTTR in buffer and human serum.
    Figure 2: TLHE1 and its peptide conjugates bind selectively to hTTR in buffer and human serum.

    (a) Chemical structure of TLHE1 and TLHE2 and four TLHE1-peptide conjugates; 5 is TLHE1 conjugated to the fluorogenic tripeptide Arg-Gly-Lys-MCA. Stability of 5 was evaluated in the in vitro trypsin assay in the presence of hTTR. 6 is TLHE1 conjugated to the N terminus of neurotensin (NT). Stability of 6 was evaluated in the human serum protease assay. 7 is TLHE1 conjugated to the N terminus of native GnRH. Stability of 7 was evaluated in the human serum protease assay and its pharmacokinetic properties were evaluated in vivo in rats. 8 is TLHE1 conjugated to the ε-amino group of Lys6 in the GnRH agonist, GnRH-A. Pharmacokinetic properties and efficacy of 8 were evaluated in vivo in rats. (b) SPR sensograms showing concentration-dependent (30–1,000 nM) binding of TLHE1 (Kd = 42 ± 5 nM) to hTTR immobilized on a sensor chip. Normalized μRiUs are plotted over time (residual s.d. = 0.8) with inset showing equilibrium binding analysis. (c) Fluorescence change caused by modification of hTTR in human serum (hTTR concentration, ~5 μM) by covalent probe monitored for 6 h in the presence of covalent probe alone (black circles) or covalent probe and hTTR ligands (colors; 10 μM). The lower the binding and fluorescence of covalent probe, the higher binding selectivity of ligand to hTTR. Each bar shows the mean ± s.d. of three replicates.

  3. Binding to TTR increased the stability of TLHE1-peptides 5, 6 and 7 in vitro, and extended the t1/2 of 7 in rats.
    Figure 3: Binding to TTR increased the stability of TLHE1–peptides 5, 6 and 7 in vitro, and extended the t1/2 of 7 in rats.

    (a) hTTR protected 5 against trypsin hydrolysis in buffer Proteolysis of Arg-Gly-Lys-MCA and 5 (10 μM) by trypsin in buffer in presence and absence of hTTR (10 μM) or AG10 (20 μM). The mixture was incubated at 37 °C for 30 min, and the proteolytic release of 7-amino-4-methylcoumarin (7-AMC) was evaluated by measuring the 7-AMC fluorescence (excitation wavelength of 345 nm and emission wavelength of 440 nm). AFU, arbitrary fluorescence units. Each bar shows the mean ± s.d. of four replicates. (b,c) hTTR protected 6 (b) and 7 (c) against proteolytic hydrolysis in human serum (hTTR concentration ~5 μM). Test compounds (5 μM) were added to serum and to serum preincubated with AG10 (10 μM). The amounts of compounds remaining in serum were quantitated at indicated time points. Each point shows the mean ± s.d. of three replicates. (d) Evaluating the pharmacokinetic profile of 7 in rats. Equivalent amounts of GnRH and 7 were administered at time 0 (i.v. bolus; 3.3 μmole/kg of each compound) to two groups of male rats (n = 4 for each group); one group was pretreated with vehicle (untreated), and the other group was pretreated with AG10 (AG10-treated group; 17.1 μmole/kg, intravenously). The concentration of test compounds in plasma was determined using a validated HPLC method (Supplementary Note 2) and plotted as a function of time after dosing. Concentrations are expressed as mean ± s.e.m. of four biological replicates.

  4. Compound 8 preferentially binds to GnRH-R over hTTR and does not interfere with holo-RBP-TTR interaction.
    Figure 4: Compound 8 preferentially binds to GnRH-R over hTTR and does not interfere with holo-RBP-TTR interaction.

    (a) SPR sensogram showing the effect of GnRH-R on 8 interaction with hTTR. Buffer (orange); GnRH-R (black, 6 nM); 8 (blue, 240 nM); 8 + GnRH-R (red, 240 nM + 6 nM); 8 + GnRH-R + GnRH-A (green, 240 nM + 6 nM + 5 μM). (b) Effect of increasing concentrations of GnRH-R on 8 (240 nM) binding to hTTR. Each bar shows the mean ± s.e.m. of three replicates (c) Modeled complex illustrating binding of 8 to hTTR and GnRH-R. hTTR and GNRH-R are too close to each other to simultaneously bind to 8 (separated by only ~3 Å). The 3D structure of GnRH-R was modeled against human δ-opioid 7TM receptor. (d) Human serum was incubated with DMSO, 8 (20 μM), or T4 (20 μM) in PBS buffer (pH 7) or with urea (8 M) buffer for 2 h at 37 °C before cross-linking and immunoblotting. The membrane was incubated with rabbit anti-RBP antibody and then with IRdye800 donkey anti-rabbit secondary antibody. Full-length gel is shown in Supplementary Figure 9. The gel is a representation of replicate experiment. (e) SPR sensogram showing that interaction of 8 + holo-RBP (4 μM and 0.2 μM, respectively; 74 ± 1 μRiU) with hTTR on sensor chip is almost a combination of individual responses to 8 (4 μM; 34 ± 0.1 μRiU) and holo-RBP (0.2 μM; 46 ± 0.1 μRiU). Normalized μRiUs are plotted over time. The sensogram is a representation of a duplicate experiment.

  5. Compound 8 displayed extended t1/2 and superior efficacy in rats.
    Figure 5: Compound 8 displayed extended t1/2 and superior efficacy in rats.

    (a) Evaluation of the pharmacokinetic properties of 8 in rats. Equivalent amounts of GnRH-A and 8 were administered at time 0 (single intravenous (i.v.) bolus; 3.3 μmole/kg of each compound) to two groups of male rats (n = 3 for each group); one group was pretreated with vehicle (untreated) and the other group was pretreated with AG10 (AG10-treated group; 17.1 μmole/kg, i.v.). The concentration of test compounds in plasma was determined using validated HPLC method and plotted as a function of time after dosing. Concentrations are expressed as means ± s.e.m. of three biological replicates. (b) Evaluating the efficacy of 8 in rats. Administration of 8 (single i.v. bolus; 225 ng/kg, 120 pmol/kg) to gonad-intact male rats (n = 4) stimulated the release of testosterone and maintains higher levels of testosterone in circulation compared to administration of equivalent dose of GnRH-A (single i.v. dose; 150 ng/kg, 120 pmol/kg) to a second group of rats (n = 4). As a control, a third group of rats (n = 3) was administered only vehicle. Testosterone levels in serum were determined using ELISA, and concentrations were expressed as means ± s.e.m. of four biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; one-way ANOVA followed by post-hoc Dunnett's multiple comparisons test.

Compounds

17 compounds View all compounds
  1. 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid
    Compound 1 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid
  2. 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 2 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  3. 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-5-(pent-4-yn-1-yloxy)benzoic acid
    Compound 3 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-5-(pent-4-yn-1-yloxy)benzoic acid
  4. 3-(3-(1-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 4 3-(3-(1-(2-(2-(2-(2-carboxyethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  5. 3-(3-(1-((14S,20S)-24-amino-14-(3-guanidinopropyl)-20-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-12,15,18-trioxo-3,6,9-trioxa-13,16,19-triazatetracosyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 5 3-(3-(1-((14S,20S)-24-amino-14-(3-guanidinopropyl)-20-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-12,15,18-trioxo-3,6,9-trioxa-13,16,19-triazatetracosyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  6. 3-(3-(1-((14S,17S,20S,23S,26S,29S)-33-amino-26-(2-amino-2-oxoethyl)-29-((S)-2-(((S)-1-(((S)-1-((S)-2-(((S)-1-(((2S,3S)-1-(((S)-1-carboxy-3-methylbutyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamoyl)pyrrolidin-1-yl)-5-guanidino-1-oxopentan-2-yl)amino)-5-guanidino-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-14-(2-carboxyethyl)-23-(carboxymethyl)-20-(4-hydroxybenzyl)-17-isobutyl-12,15,18,21,24,27-hexaoxo-3,6,9-trioxa-13,16,19,22,25,28-hexaazatritriacontyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 6 3-(3-(1-((14S,17S,20S,23S,26S,29S)-33-amino-26-(2-amino-2-oxoethyl)-29-((S)-2-(((S)-1-(((S)-1-((S)-2-(((S)-1-(((2S,3S)-1-(((S)-1-carboxy-3-methylbutyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamoyl)pyrrolidin-1-yl)-5-guanidino-1-oxopentan-2-yl)amino)-5-guanidino-1-oxopentan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-14-(2-carboxyethyl)-23-(carboxymethyl)-20-(4-hydroxybenzyl)-17-isobutyl-12,15,18,21,24,27-hexaoxo-3,6,9-trioxa-13,16,19,22,25,28-hexaazatritriacontyl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  7. (9S,15S,18S,21S,24S,27S)-24-((1H-imidazol-4-yl)methyl)-21-((1H-indol-3-yl)methyl)-1-amino-27-(3-(2-(2-(2-(4-(3-(3-carboxy-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)phenoxy)propyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-15-(4-hydroxybenzyl)-18-(hydroxymethyl)-1-imino-9-isobutyl-8,11,14,17,20,23,26-heptaoxo-2,7,10,13,16,19,22,25-octaazatriacontan-30-oic acid
    Compound 7 (9S,15S,18S,21S,24S,27S)-24-((1H-imidazol-4-yl)methyl)-21-((1H-indol-3-yl)methyl)-1-amino-27-(3-(2-(2-(2-(4-(3-(3-carboxy-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)phenoxy)propyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)propanamido)-15-(4-hydroxybenzyl)-18-(hydroxymethyl)-1-imino-9-isobutyl-8,11,14,17,20,23,26-heptaoxo-2,7,10,13,16,19,22,25-octaazatriacontan-30-oic acid
  8. 3-(3-(1-((3S,6S,9S,12S,15R)-3-((1H-imidazol-4-yl)methyl)-6-((1H-indol-3-yl)methyl)-15-(((S)-1-(((S)-6-guanidino-2-oxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)-12-(4-hydroxybenzyl)-9-(hydroxymethyl)-1,4,7,10,13,21-hexaoxo-1-((S)-5-oxopyrrolidin-2-yl)-24,27,30-trioxa-2,5,8,11,14,20-hexaazadotriacontan-32-yl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 8 3-(3-(1-((3S,6S,9S,12S,15R)-3-((1H-imidazol-4-yl)methyl)-6-((1H-indol-3-yl)methyl)-15-(((S)-1-(((S)-6-guanidino-2-oxohexan-3-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)-12-(4-hydroxybenzyl)-9-(hydroxymethyl)-1,4,7,10,13,21-hexaoxo-1-((S)-5-oxopyrrolidin-2-yl)-24,27,30-trioxa-2,5,8,11,14,20-hexaazadotriacontan-32-yl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  9. methyl 3,5-dihydroxybenzoate
    Compound 9 methyl 3,5-dihydroxybenzoate
  10. methyl 3-hydroxy-5-(pent-4-yn-1-yloxy)benzoate
    Compound 10 methyl 3-hydroxy-5-(pent-4-yn-1-yloxy)benzoate
  11. methyl 3-(3-bromopropoxy)-5-(pent-4-yn-1-yloxy)benzoate
    Compound 11 methyl 3-(3-bromopropoxy)-5-(pent-4-yn-1-yloxy)benzoate
  12. methyl 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-5-(pent-4-yn-1-yloxy)benzoate
    Compound 12 methyl 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-5-(pent-4-yn-1-yloxy)benzoate
  13. (2S,8S)-21-azido-2-(4-((tert-butoxycarbonyl)amino)butyl)-4,7,10-trioxo-8-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-13,16,19-trioxa-3,6,9-triazahenicosan-1-oic acid
    Compound 13 (2S,8S)-21-azido-2-(4-((tert-butoxycarbonyl)amino)butyl)-4,7,10-trioxo-8-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-13,16,19-trioxa-3,6,9-triazahenicosan-1-oic acid
  14. tert-butyl ((14S,20S)-1-azido-20-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-12,15,18-trioxo-14-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-3,6,9-trioxa-13,16,19-triazatetracosan-24-yl)carbamate
    Compound 14 tert-butyl ((14S,20S)-1-azido-20-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-12,15,18-trioxo-14-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-3,6,9-trioxa-13,16,19-triazatetracosan-24-yl)carbamate
  15. 3-(3-(1-((10S,16S)-2,2-dimethyl-10-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-4,12,15,18-tetraoxo-16-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-3,21,24,27-tetraoxa-5,11,14,17-tetraazanonacosan-29-yl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
    Compound 15 3-(3-(1-((10S,16S)-2,2-dimethyl-10-((4-methyl-2-oxo-2H-chromen-7-yl)carbamoyl)-4,12,15,18-tetraoxo-16-(3-(3-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)guanidino)propyl)-3,21,24,27-tetraoxa-5,11,14,17-tetraazanonacosan-29-yl)-1H-1,2,3-triazol-4-yl)propoxy)-5-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)benzoic acid
  16. 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid
    Compound 16 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoic acid
  17. 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate
    Compound 17 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)propanoate

Accession codes

Referenced accessions

Protein Data Bank

References

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Author information

  1. These authors contributed equally to this work.

    • Sravan C Penchala &
    • Mark R Miller

Affiliations

  1. Department of Pharmaceutics & Medicinal Chemistry, Thomas J. Long School of Pharmacy & Health Sciences, University of the Pacific, Stockton, California, USA.

    • Sravan C Penchala,
    • Mark R Miller,
    • Arindom Pal,
    • Jin Dong,
    • Nikhil R Madadi,
    • Jinghang Xie,
    • Trever Cox,
    • Jesse Miles,
    • William K Chan,
    • Miki S Park &
    • Mamoun M Alhamadsheh
  2. Department of Chemistry, University of the Pacific, Stockton, California, USA.

    • Hyun Joo,
    • Jerry Tsai,
    • Patrick Batoon,
    • Vyacheslav Samoshin &
    • Andreas Franz

Contributions

M.M.A. conceived and supervised the project. M.M.A, S.C.P. and M.R.M. designed the experiments. S.C.P., M.R.M. and A.P. performed the chemical synthesis, PK studies and biological assays. H.J. and J.T. performed the modeling study. J.D. and P.B. helped with mass spectrometry analysis. N.R.M., V.S. and A.F. did the NMR spectrometry analysis. J.X. and W.K.C. did the western blot assay and provided advice. M.S.P. designed and analyzed the PK studies. T.C. and J.M. helped with chemical synthesis. M.M.A., S.C.P. and M.R.M. wrote the manuscript, and all authors refined the manuscript.

Competing financial interests

The co-authors have filed a provisional patent application related to this work.

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Supplementary information

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  1. Supplementary Text and Figures (3,842 KB)

    Supplementary Results, Supplementary Table 1, Supplementary Figures 1–10 and Supplementary Notes 1 and 2

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