The s. The variant with the best affinity for albumin was selected for the next cycle of amino acid substitution and affinity testing. If several amino substitutions showed a similar affinity improvement, a change to D or E was preferred as a charged amino acid could enhance the solubility.
All results being equal, E was chosen due to the propensity of D to isomerize to iso-Asp or to racemize to D-Asp. If different positions provided similar affinity improvements, the one closest to the lysine was fixed in order to evolve the peptide from the central palmitoyl anchor to the peptide ends.
The iterative process of tag synthesis and affinity screening was repeated until optimal residues were found for all serine positions. For each cycle, the sequence of the starting peptide for example, F -S 1 S 2 S 3 K palm S 5 S 6 S 7 is shown together with its dissociation constant K d for human albumin.
One serine was substituted at the time to the indicated amino acids W, Y, L, D, E and G and the affinity of the different heptapeptide-palmitoyl variants are indicated in the boxes. Dark blue or red shaded boxes indicate variants with the best binding affinities. Variants in boxes highlighted in red were used for the next evolution cycle.
Average values of at least three measurements and s. In the first cycle of synthesis and screening, six out of the 36 tag variants, all with D or E amino acid replacements, showed an approximately twofold improvement in binding affinity greater than twofold improved K d s are highlighted in dark blue in Fig. We fixed E in position five fixed substitutions carried through to further cycles are highlighted in red in Fig.
We fixed E in position three and varied the remaining four serine positions. We followed two paths for further optimizations, one by fixing E in position two cycle 4a and one by fixing Y in position two cycle 4b. Following path 4a, no large improvements could be achieved. In contrast, path 4b enhanced the binding affinity by nearly another factor two when any of the three serine positions were occupied by E.
We subsequently followed three paths cycles 5a—c and substituted the remaining two serine positions to Y or E. In the last serine position, Y gave a slightly better affinity than E. The evolved amino acid sequence did not share any homology with binding regions of natural albumin-binding proteins. The affinities of the best tag variants from each cycle were measured on the same assay plate using the same dilutions of albumin to exclude inaccuracies rising from the separate preparation of albumin solutions Fig.
The graphic shows that the binding was improved by around a factor two in the first rounds of evolution, and that affinity enhancements decreased in the last cycles as we sharpened the affinity to the final peptide sequence. Transferring the fluorescein in the final peptide EYEK palm EYE from the N- to the C-terminus reduced the binding affinity several fold, which suggested that the fluorescein moiety is participating in the binding to albumin when attached to the N-terminus.
In blood, fatty acids bound to albumin could potentially compete with the binding of the palmitoylated heptapeptide. Incubation of the evolved tag and best variants with human serum showed efficient binding in the context of fatty acids and other blood components Fig. To apply the tag in pre-clinical studies, it is necessary that it binds to the albumin of other species.
Analysis of the best tag variants in the same assay showed that the binding to rat albumin was also improved in each evolution cycle but to a smaller extent Fig. Residual inhibition activities were quantified in protease activity assays using fluorogenic substrates of plasma kallikrein and urokinase. Both protease inhibitors suffered from fast renal clearance upon previous tests in mice Fluorescein was included in the tag for binding purposes as well as for the facile measurement of the K d by fluorescence polarization and the detection of the tagged peptides in blood samples.
The two conjugates showed a solubility in PBS of 1. As expected, a control with non-tagged UK18 showed no binding to albumin.
As with the tag alone, the tagged bicyclic peptides displayed a similar affinity to human albumin when measured in serum compared to the purified protein Table 1. The binding of the tagged bicyclic peptides to rat albumin decreased three to fourfold compared to the tag alone and was overall around fivefold weaker than the binding to human albumin Table 1.
Both of the two tagged bicyclic peptides inhibited the protease targets with K i s in the nanomolar range, tag-PK plasma kallikrein with a K i of 9. The inhibitory activity of tagged PK was thus fold weaker and that of tagged UK18 was unchanged. It is likely that the tag sterically hinders binding of PK to plasma kallikrein to some extent and that this effect is enhanced when it is bound to albumin. We tested the half-life of tag-PK and tag-UK18 in rats by i. For comparison, we measured the half-life of UK18 0.
The tagged bicyclic peptides had elimination half-lives of 2. The tag alone had a longer elimination half-life Detailed pharmacokinetic parameters including distribution half-life, clearance rate and volume of distribution are shown in Table 2. For the conjugates tag-PK and tag-UK18, no degradation products could be detected, which again indicates the protective effect of the conjugation Supplementary Figs 9 and Despite its linear format, the tag alone was not degraded, most likely because all amino acids of the peptide interact with albumin or are near its surface and thus are not accessible to proteases Supplementary Fig.
Several recent studies indicate that coagulation factor XII FXII plays a key role in various thrombotic disorders and have featured the serine protease as a therapeutic target For application in antithrombotic therapy, the inhibitor needs to block FXIIa over an extended period of time. To enable prolonged exposure, we applied the tag to the FXIIa inhibitor. Conjugation of the tag to either end of the bicyclic peptide reduced its affinity over fold wherein PEG linkers of various lengths attached between the tag and inhibitor limited the loss in affinity somewhat Supplementary Table 1 , Supplementary Figs 14— Inhibition of FXIIa in blood can be assessed by measuring the so-called activated partial thromboplastin time, the time until citrated plasma clots upon activation of the intrinsic coagulation pathway.
We measured this parameter in rabbit plasma ex vivo. The conjugate bound rabbit albumin with a K d of 1. Upon i. Concentration of FXIIa inhibitor in rabbit plasma after i. Lipidation is an established strategy for extending the circulation time of peptide drugs as showcased by the approved drugs insulin detemir, insulin degludec and liraglutide. In all of these peptide drugs, fatty acids such as myristic or palmitic acid are appended to an amino acid within the peptide sequence itself.
It can be expected that the plasma half-life of these or other peptide drugs could be substantially prolonged by applying the concept of embedding the fatty acid into a peptide sequence that enhances the binding to albumin. Studies on fatty acid-modified insulin analogues and peptide-based albumin binders showed that a stronger affinity for albumin correlated with a prolonged half-life 27 , Herein, we demonstrate that a peptide sequence as short as seven amino acids including the lipidated lysine can improve the binding of a fatty acid conjugated to a peptide by fold.
The peptide was evolved with a simple and efficient strategy based on six rounds of iterative amino acid substitution and affinity measurement, in which more than heptapeptide-palmitoyl tags were tested. As expected, amino acids with negatively charged or hydrophobic side chains were essential for the increased binding affinity, although the specific sequence of these amino acids was also an important factor. Due to the four negative charges in the peptide, the tagged conjugates have a good solubility in aqueous solution, despite the long hydrophobic tail of palmitic acid, preventing solubility issues of the lipidated peptides.
We showed that the tag can be appended conveniently to cargo peptides by automated synthesis on a standard peptide synthesizer. Application of the tag to a bicyclic peptide FXIIa inhibitor allowed efficient blockage of the intrinsic coagulation pathway in rabbits as long as eight hours. It is important to note that the tag has a weaker affinity for rat and rabbit than for human albumin five to sevenfold.
Taking this difference and allometric scaling considerations into account, it is likely that peptide drugs modified with the tag can achieve half-lives of more than one day in human. If such conjugates are administrated as a depot, an even longer exposure may be reached. With this tag in hand, it should be possible to expand the application range of peptide therapeutics from the current mostly short-lived agents that act mainly as receptor agonists to long-acting peptide drugs that can also address targets that require actions over extended time periods, such as the inhibition of enzymes or receptors.
The coupling was carried out twice for each natural amino acid 4 equiv. The coupling of unnatural amino acids 2 equiv. Washing steps were performed with DMF. Therefore, the N-terminus was protected by Boc in peptides not modified with fluorescein.
Peptides were acylated at the lysine side chain by the addition of free fatty acid 2 equiv. Finally, peptides were either lyophilized or directly purified. Fractions containing the desired peptide were lyophilized.
The mass of purified peptides was determined by electrospray ionization mass spectrometry ESI-MS in positive ion mode on a single quadrupole liquid chromatograph mass spectrometer LCMS, Shimadzu. Human albumin A , rat albumin A , rabbit albumin A and human serum H were purchased from Sigma-Aldrich. Dissociation constant K D were determined by nonlinear regression analysis of anisotropy versus the total concentration of proteins [P] T using the following equation:.
A is the experimental fluorescence anisotropy. A f and A b are the fluorescence anisotropy for the free and the fully bound fluorescent ligand, respectively.
K D is the dissociation constant for the binding equilibrium. Values were calculated with Prism 5 GraphPad software. This process was repeated once.
K i values of UK18 and tagged UK18 were determined by measuring residual activity of recombinant human urokinase 19 1. K i values of PK and tagged PK were determined by measuring residual activity of human plasma kallikrein 0. Sigmoidal curves were fitted to the data using the following four-parameter logistic equation used for dose—response curves. IC 50 values were derived from the fitted curve using Prism 5 GraphPad software. The specificity of tagged FXIIa inhibitor was assessed by measuring the inhibition of the following human serine proteases: urokinase 1.
Enzymes were purchased from Molecular Innovations or Innovative Research. The inhibitory activity of each sample was assessed by preparing a series of twofold dilutions and measuring residual protease activity for urokinase and plasma kallikrein as described above. The resulting percentages of inhibition were plotted in Prism 5 software GraphPad software versus time and analysed using a one-phase decay model.
Female Sprague Dawley rats were injected with 0. Peptide injection and blood sample collection was performed by Washington Biotech Inc. HA is mainly catabolised in the muscles, liver and kidneys. Serum albumins are important in regulating blood volume by maintaining the oncotic pressure of the blood compartment and maintaining fluid balance in the body. They also serve as carriers for poorly water soluble molecules including hormones, bile salts, unconjugated bilirubin, free fatty acids, ions, and some drugs, with important consequences for their solubilisation, transport, metabolism, and detoxification.
Search: Search. Home » Peptide Family » Albumin peptides. Albumin peptides. Furthermore, we have recently shown that the shift of plasma albumin redox state to an oxidized state in growing rats, induced by ingestion of a casein CN -based low protein LP diet, was ameliorated when cystine Cyss was added to the CN-based LP diet As sulfur amino acids are limiting amino acids for growth and maintenance of rats when they are placed on CN as a protein source 17 , it was considered that the redox state of plasma albumin would be shifted to a more oxidized state by dietary amino acid imbalance such as shortage of Cys in CN.
Thus, it was suggested that plasma albumin redox state could also be associated with the quality of dietary protein such as amino acid balance. However, the above recent study of ours was conducted only in a short period of time 2 weeks , and energy and protein intakes differed significantly between some of the dietary groups A longer-term study with the feeding of equal amount of protein sources is likely necessary to substantiate the association between protein quality and plasma albumin redox state.
The aim of this study was to elucidate the role of protein quality in determining the redox state of plasma albumin, by focusing on amino acid balance in diet. Growing rats were subjected to three kinds of dietary experiments based on our previous study 14 , where animals fed low protein diet initially manifested a decrease in plasma MA ratio and then exhibited hypoalbuminemia. It was therefore considered that this animal model clearly expresses the progression of protein undernutrition could be useful to indicate the difference in the quality of dietary proteins.
In Experiment 1, effects of protein sources with different amino acid compositions on the redox state of plasma albumin was investigated under a pair-feeding regimen, such as CN, whey protein WP , or wheat gluten WG. CN and WP are both of milk origin. They differ significantly in amino acid balance and gastrointestinal digestive motility 18 , 19 , while both have been reported to have excellent protein digestibility as assessed by ileal digestibility in pigs and rats 20 — In Experiment 2, the role of limiting amino acids in determining plasma albumin redox state was scrutinized, by placing animals initially on a CN-based low protein diet without Cyss supplementation, and subsequently maintaining them on the diet supplemented with Cyss; we examined whether Cyss supplementation during the last 2 weeks would improve dietary amino acid balance, thereby alleviating oxidized shift of plasma albumin.
In Experiment 3, effects of dietary Cys were further explored by supplementing it in the form other than Cyss. After 1 week of acclimation, animals were subjected to 3 kinds of experiments. These 5 dietary groups were maintained for 4 weeks. After 4 weeks of the above dietary regimen, animals were euthanized by deep anesthesia with sevoflurane Mylan, Canonsburg, PA.
Blood was drawn from the inferior vena cava, and plasma layers were obtained after centrifugation. Liver samples were also excised and frozen immediately in liquid nitrogen.
These 6 dietary groups were fed ad libitum for 4 weeks, and were then euthanized. Blood was obtained by cardiac puncture, and plasma layers were obtained after centrifugation.
Plasma samples were applied to an amino acid analyzer L; Hitachi High-Technologies, Tokyo, Japan to analyze free amino acid patterns, as described previously 14 , The redox state of plasma albumin was determined as described previously 14 , Fluorescence emission was measured at nm for excitation and nm for emission. Animals were placed on one of these LP diets under a pair-feeding regimen for 4 weeks.
Energy and protein intakes were significantly higher in the CT diet group than the LP diet groups, and no significant difference was seen between the LP diet groups Table 2. Body weight of the CT group at 4 weeks was significantly higher than those of the LP diet groups, and the levels did not differ significantly between the LP diet groups Table 2.
Table 2. Energy intake, protein intake, body weight, and plasma free amino acid levels in Experiment 1. Patterns of free amino acid were analyzed for plasma samples obtained at the end of the experimental period, as plasma free amino acid levels reflect immediate intakes of dietary proteins and amino acids Both total and essential amino acid levels were lower in all the LP diet groups compared with the CT diet groups, and most of the differences were statistically significant Table 2.
There were also significant differences in these levels between some of the LP diet groups, but they differed to limited extents. The levels of albumin were determined for plasma samples obtained during the experimental period. Plasma albumin levels in all the LP diet groups initially decreased, and remained constant thereafter Figure 1.
The decreased plasma albumin levels in the LP diet groups were significantly lower than the levels of CT diet group. Figure 1. Plasma albumin levels in Experiment 1. The levels of albumin were measured for plasma samples obtained during the experimental period. Plasma samples, obtained during the experimental period, were analyzed to determine the albumin redox state Figure 2.
As was seen in our previous studies 13 , 14 , 16 , plasma albumin redox state shifted to a more oxidized states in all the LP diet groups. When it was expressed as the ratio of MA among total albumin, the ratio in the LP diet groups dropped rapidly early in the experimental period Figure 3.
Figure 2. Chromatograms of plasma albumin in Experiment 1. Plasma samples, obtained during the experimental period, were subjected to HPLC to determine albumin redox state. MA and NA denote mercaptalbumin and non-mercaptalbumin, respectively. Figure 3. Ratio of mercaptalbumin among total plasma albumin in Experiment 1. Plasma samples, obtained weekly, were subjected to HPLC to determine albumin redox state.
As albumin synthesis is primarily regulated at the transcription level 2 , albumin gene expression in livers was examined at the end of the experimental period. Albumin expression was significantly suppressed in the LP diet groups compared with the CT diet group Figure 4. Figure 4. Hepatic albumin and 4E-BP1 gene expression in Experiment 1.
Gene expression of albumin and eukaryotic initiation factor 4E-binding protein 1 4E-BP1 was determined in livers obtained at the end of the experimental period. Our recent study showed that 4E-BP1, an eukaryotic translation initiation factor, was induced at both transcriptional and translational levels in the livers of young rats placed on a CN-based LP diet, thereby suppressing downstream protein synthesis including albumin; lower Cys content in CN is likely responsible for the 4E-BP1 induction As MA ratio correlates with albumin synthesis rate 14 , it is considered that this Cys-mediated regulatory system could also modulate the MA ratio of the LP diet groups.
Compared with the CT diet group. However, no significant difference in this gene expression was seen between the LP diet groups. Table 3. Energy intake, protein intake, body weight, and plasma free amino acid levels in Experiment 2.
Free amino acid patterns of plasma samples, obtained at the end of the experimental period at week 6 , were analyzed. Neither total amino acid levels nor essential amino acid levels were significantly different between the two LP diet groups Table 3.
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