Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum

<div><p>Proteolytic degradation of peptide-based drugs is often considered as major weakness limiting systemic therapeutic applications. Therefore, huge efforts are typically devoted to stabilize sequences against proteases present in serum or plasma, obtained as supernatants after complete blood coagulation or centrifugation of blood supplemented with anticoagulants, respectively. Plasma and serum are reproducibly obtained from animals and humans allowing consistent for clinical analyses and research applications. However, the spectrum of active or activated proteases appears to vary depending on the activation of proteases and cofactors during coagulation (serum) or inhibition of such enzymes by anticoagulants (plasma), such as EDTA (metallo- and Ca²⁺-dependent proteases) and heparin (e.g. thrombin, factor Xa). Here, we studied the presumed effects on peptide degradation by taking blood via cardiac puncture of CD-1 mice using a syringe containing a peptide solution. Due to absence of coagulation activators (e.g. glass surfaces and damaged cells), visible blood clotting was prevented allowing to study peptide degradation for one hour. The remaining peptide was quantified and the degradation products were identified using mass spectrometry. When the degradation rates (half-life times) were compared to serum derived freshly from the same animal and commercial serum and plasma samples, peptides of three different families showed indeed considerably different stabilities. Generally, peptides were faster degraded in serum than in plasma, but surprisingly all peptides were more stable in fresh blood and the order of degradation rates among the peptides varied among the six different incubation experiments. This indicates, that proteolytic degradation of peptide-based therapeutics may often be misleading stimulating efforts to stabilize peptides at degradation sites relevant only <i>in vitro</i>, i.e., for serum or plasma stability assays, but of lower importance <i>in vivo</i>.</p></div

Stability of eight peptides in fresh blood, serum, and plasma.

<p>Stability of eight peptides in fresh blood, serum, and plasma.</p

Degradation of oncocin derivatives.

<p>Onc18, Onc72, and Onc112 were analyzed after one hour incubation in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue). Peptides were separated by RP-HPLC in the presence of 0.1% trifluoroacetic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero.</p

Fibrinopeptide A and its degradation products.

<p>Peak areas obtained from extracted ion chromatograms of triply protonated murine fibrinopeptide A (DTEDKGEFLSEGGGVR, purple; <i>m/z</i> 565.9) and its triply protonated degradation products TEDKGEFLSEGGGVR (green; <i>m/z</i> 527.6) and EDKGEFLSEGGGVR (white; <i>m/z</i> 493.9). Six to nine samples were analyzed for each matrix (blood, B; direct serum, DS; activated serum, AS) and time point.</p

Degradation of apidaecin derivatives.

<p>Peptides in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue) were analyzed after 10 min (Api88) and one hour (Api134 and Api137) incubation. Separation was performed by RP-HPLC in the presence of 0.1% trifluoroacetic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero.</p

Sequences of all degradation products identified in blood, serum, and plasma samples.

<p>Sequences of all degradation products identified in blood, serum, and plasma samples.</p

Degradation of elongated Onc112 derivatives.

<p>AAYR-Onc112 (left) and LVPR-Onc112 (right) were analyzed after one hour incubation in blood (dark red), direct serum (red), activated serum (orange), commercial serum (yellow), heparin plasma (dark blue), and EDTA plasma (blue). Peptides were separated by RP-HPLC in the presence of 0.1% formic acid and detected by absorbance at 214 nm. Peptide amounts were calculated relative to the quantities determined at time point zero. Onc112 (white) was released from both constructs, whereas metabolites YR-Onc112 (squared) and R-Onc112 (striped) were detected only for AAYR-Onc112.</p

Sequences and monoisotopic masses of all studied peptides.

<p>Sequences and monoisotopic masses of all studied peptides.</p

Identification of Api88 Binding Partners in <i>Escherichia coli</i> Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with <i>p</i>-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. <i>Escherichia coli</i> was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism

Identification of Api88 Binding Partners in <i>Escherichia coli</i> Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with <i>p</i>-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. <i>Escherichia coli</i> was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism