The major limitation for using peptides as affinity reagents, probes and therapeutics is their inherent instability in biological environments. Although the amide backbone is chemically stable, peptides are readily broken down in a matter of seconds in the digestive tract, in blood, plasma, serum and inside cells due to the presence of proteases. Because of this instability, many routes have been devised to chemically alter or modify natural peptides such as 1) including the addition of N-methylation to the backbone, 2) insertion of β-amino acids, 3) changing the location of the side chain (e.g., peptoids), or 4) by covalent cyclization via insertion of chemical bridges.
Left unanswered by these studies is the question of how much a functional peptide with natural amino acids could be stabilized by sequence optimization alone. Previously, we demonstrated a route to create high-diversity cyclic peptide libraries via mRNA display and used this approach to isolate a high affinity binder termed cycGiBP to the signaling protein Gαi1-GDP. cycGiBP showed a very high affinity (K d = 2.1 nM) and ~3-fold increase in protease resistance as compared with the corresponding linear sequence. One observation in that work was that only one of the three possible cleavage sites predominated when cycGiBP was subjected to chymotrypsin. Using the standard substrate notation for proteases (P 3-P 2-P 1-P 1′-P 2′-P 3′; P's represent the amino acid identity at a position and the scissile bond is located between P 1 and P 1′ residue), chymotrypsin has a strong P 1 preference for W > Y > F ≫ L. However, cycGiBP shows cleavage at P 1 = Y 5, but not at P 1 = W 4 or F 7.
We wondered if there were other members in that library that had improved protease resistance while retaining binding function. To address this issue, we performed a dual selection for chymotrypsin resistance and binding function on a library previously only sieved for binding function. Those experiments resulted in a several highly protease resistant peptides and indicate that sequence optimization can improve hydrolytic stability by as much as 400-fold compared to peptides sequences isolated without this selective pressure.
Gαi1-GDP with a C-terminal BirA tag was expressed and purified as previously described. mRNA display selection targeting Gαi1-GDP was performed starting at round 7 of our previous work and performed as described with the modification that the cDNA fusions were digested with 2 mg of immobilized chymotrypsin (Sigma-Aldrich) per 10 6 cpm of fusions at room temperature for 15 minutes in 50 mM Sodium Phosphate buffer (pH = 8.0). The chymotrypsin beads were removed by centrifugation through 0.45 μm filters before the selection step.
R6A (MSQTKRLDDQLYWWEYL), Biotin-labeled R6A (Bio-MSQTKRLDDQLYWWEYL), cycPRP-1 (MITWIDFISPSK), cycPRP-2 (MTWFEYLSGSK), cycPRP-3 (MTWFEFLSSTSK) and cycGiBP (MITWYEFVAGTK) were synthesized on Rink Amide AM Resin LL (Novabiochem) and cyclized using DSG as described by Millward et al. After the reaction the cyclized peptides were purified via C 18 HPLC and the mass confirmed by MALDI-TOF MS.
Binding constants were determined relative to the R6A peptide [K d = 60 nM] by equilibrium competition using 35[S]-Met radiolabeled Gαi1-GDP and biotinylated R6A as previously described and the data analyzed using GraphPad Prism 5.0.
Peptides (250 nmol of peptide in DMSO) were added to 50 mM sodium phosphate buffer (pH 8.0) with a final DMSO concentration of 2% (v/v). Sixty units of immobilized chymotrypsin (Sigma Aldrich) were added and allowed to incubate at room temperature. Aliquots were taken at various time points and subsequently filtered. The aliquots were then injected onto a C 18 reverse phase column and separated by a gradient elution from 15 to 90% B in 25 minutes. Solvent A consisted of 0.1% (v/v) TFA in water and solvent B contained CH 3 CN with 0.1% (v/v) TFA. The area under the starting material peak was quantitated using the 32 KaratGold Software package (Beckman). The graph was generated by fitting the data to a one phase exponential decay equation (GraphPad Prism 5.0). The mean and the standard error are reported in table 1.
Table 1 Chymotrypsin resistance of linear and cyclic
The peptides were prepared and characterized as described above in the protease resistance experiment. Only a single 2 minute time point with varying concentrations of the peptides (0, 5, 11, 22, 65 and 260 μM) was analyzed using Michaelis-Menten enzyme kinetics regression equation (GraphPad Prism 5.0). The mean and the standard error are reported in table 3.
Table 2 K m and V max enhancements for GiBP and PRPs
Table 3 Stability of linear and cyclic peptides in human serum
Lyophilized human serum (Thermo Scientific) was reconstituted by adding 2 mL ddH 2 O to each 5 mg vial from the manufacturer. Each reaction contained 250 nmol of peptide, 50 μL of sodium phosphate buffer (pH 8.0) and 10% DMSO (v/v). One milliliter of reconstituted serum was added to each sample and incubated at 37°C. For each time point, 100 μL aliquots were taken from the reaction and quenched in 300 μL of acetonitrile. These quenched samples were centrifuged to separate precipitate and the supernatant was diluted in water to 1.5 mL. Samples were then analyzed via HPLC as described above. The mean and the standard error are reported in table 3.
Far UV-CD spectra were obtained using a Jasco J810 spectropolarimeter (located at the USC NanoBiophysics Core Facility) equipped with a Peltier device. The peptides (25–100 μM) were prepared in 10 mM phosphate buffer at pH 7.4 and placed in a 1 mm path length cuvette. Thereafter, CD spectra were recorded in the range of 195–240 nm. Five spectra were acquired and averaged. Spectra were baseline corrected by subtracting blank spectra of the corresponding solutions without peptide and ellipticities were converted to mean residue molar ellipticities in degrees cm 2 dmol−1.
Previously, we created a trillion-member mRNA display cyclic peptide library with the form MXXXXXXXXXK (termed MX 10 K) and used seven rounds of selection to isolate cyclic peptides that bind to the signaling protein Gαi1-GDP. The best peptide from that selection was cyclic GiBP (cycGiBP), a specific, 12-residue cyclic peptide with a K d = 2.1 nM. Additionally, we found that cycGiBP was ~3-fold more resistant to chymotrypsin as compared to a linear version of the peptide.
In an effort to see if protease-resistant, natural-sequence peptides could be found, we took the Pool 7 library from our original experiment (Figure 1a) and subjected it to a two-step selection protocol (Figure 1b). After cyclization with DSG, the library of mRNA peptide fusions was first subjected to degradation by immobilized chymotrypsin for 15 minutes at room temperature and then selected for binding to the target of interest, here Gαi1-GDP. We chose the 15 minute digestion time because preliminary experiments showed that more than 90% of the library was degraded under these conditions (data not shown) and our previous results indicated that cycGiBP should have been similarly degraded. We reasoned this incubation should thus provide sufficient selection pressure to reveal protease-resistant sequences. After three rounds of selection, representative clones from Pool 10 were sequenced (Figure 1c).
Figure 1
mRNA Display selection for chymotrypsin resistance.
(a) Pool 7 of the cyclic peptide library (MX 10 K) targeting Gαi1-GDP was used as the starting point for the selection. (b) In rounds 8–10, the library was cyclized and subjected to chymotrypsin degradation and binding selection. (c) Representative clones from Pool 10 were sequenced (see supplemental information) and peptides cycPRP-1, cycPRP-2 and cycPRP-3 were further characterized.
We then chose three sequences from pool 10 for further characterization. We term these molecules cycPRPs for Cyclic Protease Resistant Peptides (cycPRP-1, cycPRP-2 and cycPRP-3). The cycPRP sequence consensus in Pool 10 is relatively similar to the consensus sequence seen in Pool 7 and also in other Gαi1-GDP binding peptides. The core sequence of the cycPRPs (TWIDFI, TWFEYL, TWFEFL) is very similar to cycGiBP (TWYEFV), R6A (YWWEYL), KB-752 (TWYDFL).
