Cyclic β2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease

Abstract Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties.


Introduction
Natural translation machinery only utilizes the 20 proteinogenic α-amino acids (pAAs) as building blocks for protein (peptide) synthesis.However, genetic code reprogramming enables us to ribosomally synthesize peptides containing various nonproteinogenic amino acids (npAAs), including β-amino acids, in place of pAAs. 1,2To facilitate reprogramming, we have developed the flexible in vitro translation (FIT) system, which consists of precharged npAA-tRNAs prepared by using flexizymes and a reconstituted Escherichia coli translation system. 3,46][7][8] In particular, the incorporation of multiple/consecutive β-amino acids has been extremely inefficient compared with that of a single β-amino acid.
The difficulties of incorporating β-amino acids can be attributed to the following 2 reasons: (1) slow accommodation of npAA-tRNA onto the ribosomal A-site mediated by elongation factor thermo unstable (EF-Tu) and ( 2) slow peptidyl transfer of the P-site peptidyl-tRNA onto the A-site npAA-tRNA catalyzed by the peptidyl transferase center of the ribosome. 9The slow accommodation and slow peptidyl transfer can induce ribosomal stalling and mistranslocation of peptidyl-tRNA, resulting in peptidyl-tRNA drop-off.To accelerate the accommodation of npAA-tRNA (to address issue [1]), we engineered tRNA to be efficiently recognized by EF-Tu.Since the binding of aminoacyl-tRNA to EF-Tu is regulated by the T-stem region of tRNA, we designed an engineered tRNA, referred to as tRNA GluE2 , in which the T-stem was replaced by that of E. coli tRNA Glu with a high binding affinity for EF-Tu (Fig. 1a). 10he expression level of peptides containing β-amino acids was significantly improved using tRNA GluE2 compared with the use of weaker tRNAs. 11To promote peptidyl transfer between noncanonical residues (to address issue [2]), we introduced a proline-specific elongation factor, named EF-P, and optimized the D-arm region of tRNA.EF-P is known to promote peptidyl transfer reactions between consecutive prolines in nature. 12,13e have previously reported that EF-P recognizes Pro-tRNA Pro by a specific D-arm motif of tRNA Pro isoacceptors. 14We combined the T-stem and D-arm motifs into an engineered tRNA, namely tRNA Pro1E2 (Fig. 1b). 15Efficient incorporation of β-amino acids and their derivatives, including β 3 -amino acids, β2,3 -amino acids, α-aminoxy acids, and α-hydrazino acids, has been accomplished using tRNA Pro1E2 in the presence of EF-P. 11,16,17Furthermore, we have successfully incorporated consecutive β-amino acids, such as up to 7 consecutive β-homomethionines and up to 10 consecutive (1S,2S)-2-aminocyclopentane carboxylic acids, which was not attainable with ordinary suppressor tRNAs, such as tRNA AsnE2 . 7,18In addition to β-amino acids, various types of npAAs, such as D-amino acids, α,α-disubstituted amino acids, and γ-amino acids, have been efficiently incorporated using the devised translation system. 10,15,19,20he FIT system can be applied to the ribosomal synthesis of random peptide libraries comprising over 10 12 unique members with various β-amino acids.Such libraries are compatible with in vitro selection methodologies, such as mRNA display, for the discovery of bioactive peptides.2][23][24] Notably, cyclic β 2,3 -amino acids (cβAAs) (see Fig. 2a for the structures of representative cβAAs used in this study), which possess constrained cyclic structures, are interesting building blocks with defined folding propensities.][27][28][29][30][31][32][33][34][35][36][37][38][39] Recently, we performed RaPID selections using macrocyclic peptide libraries containing cβAAs and cyclizing D-α-amino acids against 2 therapeutic targets: activated coagulation factor XII (FXIIa) and interferon-gamma receptor I (IFNGR1). 16,23The resulting highly potent inhibitors prompted us to further explore macrocyclic peptide inhibitors containing cβAAs against another important therapeutic target, SARS-CoV-2 main protease (M pro ), 40,41 also known as 3-chymotrypsin-like cysteine protease (3CL pro ) and nonstructural protein 5 (nsp5), which plays an essential role in viral replication, cleaving viral polyproteins into functional proteins. 42Here we report studies aimed at obtaining macrocyclic peptides with high M pro inhibitory activity and proteolytic stability from a ribosomally synthesized macrocyclic peptide library, taking advantage of the unique folding propensities of cβAAs that contribute to enhanced binding affinity and proteolytic stability.

Preparation of tRNAs and flexizymes
tRNAs and flexizymes (dFx and eFx) were transcribed in vitro using the T7 RNA polymerase from the corresponding template DNAs prepared by using extension and the polymerase chain reaction (PCR) amplification (see Supporting Information [SI] Supplementary Table S1 for primer sequences).Extension and PCR were carried out using the following reaction mixture: 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% (v/v) Triton X-100, 2.5 mM MgCl 2 , 250 μM dNTP mix, and 60 nM Taq DNA polymerase.2 μM each of forward and reverse extension primers were added to the extension mixture and the reaction was initiated at 95 °C for 1 min followed by 5 cycles of 50 °C for 1 min and 72 °C for 1 min.The resulting solution and 0.5 μM each of forward and reverse PCR primers were added to the PCR mixture.PCR was performed for 15 cycles of 95 °C for 40 s, 55 °C for 40 s, and 72 °C for 40 s.The PCR product was purified by using phenol/chloroform extraction and ethanol precipitation.Transcription of RNA was carried out at 37 °C for 16 h using the following mixture: 40 mM Tris-HCl (pH 8.0), 22.5 mM MgCl 2 , 10 mM dithiothreitol, 1 mM spermidine, 0.01% Triton X-100, 3.75 mM (tRNAs) or 5 mM (flexizymes) nucleoside triphosphate (NTP) mix, 0.04 U/μL RNasin RNase inhibitor (Promega, N2615), and 120 nM T7 RNA polymerase.For transcription of tRNAs, 5 mM guanosine monophosphate (GMP) was added to the above solution to introduce a monophosphate at the 5′ end of the tRNAs.The resulting RNA transcripts were then treated with RQ1 DNase (Promega, M6101) at 37 °C for 30 min and purified by using 8% (tRNAs) or 12% (flexizymes) denaturing polyacrylamide gel electrophoresis (PAGE) containing 6 M urea.Miura et al.

Preparation of aminoacylated tRNAs by using flexizymes
cβAAs and D-cysteine (c) were pre-activated as their 3,5-dinitrobenzyl esters; N-chloroacetyl-D-tyrosine ( ClAc y) was activated as its cyanomethyl ester. 4,16These activated amino acids were charged onto the respective tRNAs using flexizymes (dFx for 3,5-dinitrobenzyl ester or eFx for cyanomethyl ester).Aminoacylation was carried out at 4 °C for 6 h for c, for 16 h for cβAAs, or 2 h for ClAc y in the following mixture: 600 mM MgCl 2 , 20% (v/v) dimethyl sulfoxide (DMSO), 25 μM dFx or eFx, 25 μM tRNA, and 5 mM activated amino acid.The reaction pH was adjusted by using bicine-KOH (pH 8.7) for cβAAs or HEPES-KOH (pH 7.5) for c and ClAc y.The reaction was stopped by the addition of 4× volume of 0.3 M acetate (pH 5.2) and 10× volume of ethanol.The resulting aminoacyl-tRNAs were subjected to ethanol precipitation by using centrifugation (15,000 × g, 25 ° C, 15 min) and the pellets were washed with 70% ethanol.

Production of recombinant SARS-CoV-2 M pro
Recombinant M pro was prepared as reported.Freeze/thawing cycles of M pro were avoided so as not to compromise activity. 43,44

Solid-phase peptide synthesis
Macrocyclic peptides were chemically synthesized on a milligram scale via standard Fmoc solid-phase peptide synthesis using a Syro I automated peptide synthesizer (Biotage).NovaPEG Rink Amide Resin (54 mg, 25 μmol) was incubated with N, N-dimethylformamide (DMF) at room temperature for 1 h.Each Fmoc-protected amino acid was coupled at 30 °C for 40 min on the resin in a DMF solution containing 0.2 M Fmoc-protected amino acid (6 equiv.),0.2 M 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 5 equiv.),0.2 M 1-hydroxybenzotriazole (HOBt; 5 equiv.),and 0.1 M N,N-diisopropylethylamine (DIPEA; 12 equiv.).After the resin was washed 5 times with 600 μL DMF, the Fmoc group was deprotected with 600 μL of 40% (v/v) piperidine in DMF at 30 °C for 12 min.Coupling of the Fmoc-protected amino acid and Fmoc deprotection were repeated as required.After automated peptide synthesis, 0.2 M chloroacetyl N-hydroxysuccinimide ester (8 equiv.) in N-methylpyrrolidone was added to the resin; the mixture was incubated at room temperature for 1 h with rotation.After the resin was subsequently washed with DMF 3 times and with dichloromethane 5 times, the resin-bound peptides were treated with 2 mL of a solution of 92.5% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) water, 2.5% triisopropylsilane (TIS), and 2.5% 3,6-dioxa-1,8-octanedithiol (DODT) at room temperature for 3 h with rotation to deprotect the side-chain protecting groups and to cleave the peptide off from the resin.The resulting linear peptides were precipitated with diethyl ether, then dissolved in 10 mL of 80% (v/v) DMSO, 20% (v/v) water, and 0.1% (v/v) TFA.Following the addition of 200 μL of 0.5 M tris(2carboxyethyl) phosphine (TCEP) and triethylamine to adjust the pH to 8, the peptide mixture was incubated with rotation at room temperature for 16 h to form a thioether bond between the N-terminal chloroacetamide and the thiol group of the downstream cysteine.Macrocyclization of the peptides was confirmed by using matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS); the crude peptides were purified by using reverse-phase high performance liquid chromatography (HPLC) (Shimadzu) with a Chromolith Prep RP-18 column (Merck).

Evaluation of binding affinity of peptides by using surface plasmon resonance
The binding affinities of peptides to M pro were evaluated through surface plasmon resonance (SPR) using a Biacore T200 instrument (Cytiva) at 25 °C with the following running buffer: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% (v/ v) Tween 20, and 0.1% (v/v) DMSO.Biotin-tagged M pro was immobilized on a sensor chip CAP (Cytiva) to a surface density of 1,000 to 1,500 response units following the immobilization protocols provided by Cytiva.The kinetic constant was determined using a single-cycle kinetics method by the injection of 5 different concentrations (2-fold dilution series) of each peptide at a flow rate of 30 μL/min.The resulting sensorgram was fitted to the standard 1:1 interaction model and analyzed using the Biacore evaluation software (Cytiva).

Solid-phase extraction coupled to MS inhibition assays
Inhibition of M pro was measured by solid-phase extraction (SPE) purification coupled to MS analysis using a RapidFire (RF) 365 high-throughput sampling robot (Agilent) connected to an iFunnel Agilent 6550 accurate mass quadrupole time-of-flight (Q-TOF) spectrometer as reported. 43,45In brief, the cyclic peptides were dispensed in an 11-point, 3-fold dilution series (top concentration of 25 to 5 μM) using an acoustic Echo Dispenser machine (LabCyte).Formic acid and DMSO were used as positive and negative inhibition controls, respectively.The assays were performed using isolated recombinant SARS-CoV-2 M pro (75 nM), which was prepared as reported, 43,44 and a 37-mer peptide substrate (ALNDFSNSGSDVLYQPPQTSITSAVLQ/ SGFRKMAFPS-NH 2 ; 4 µM).Reactions were incubated (15 min), then quenched by the addition of 10% (v/v) aqueous formic acid (5 μL/well).

Serum stability assays
A synthetic macrocyclic peptide (10 μM) and a serum-resistant internal standard peptide consisting only of D-amino acids (NH 2 -PEG 5 -wstndwstnd-PEG 5 -CONH 2 , 5 μM) were mixed and incubated in human serum (Cosmo Bio, 12181201) at 37 °C for up to 168 h. 16At each time point, 4 μL of the mixture was removed and quenched by adding 12 μL of methanol, followed by incubation on ice for 15 min.Following centrifugation (15,000 × g, 25 °C, 10 min), 10 μL of the supernatant was mixed with 40 μL of 1% (v/v) TFA in water.Following centrifugation (15,000 × g, 25 °C, 5 min), the supernatant was analyzed using LC/MS employing a reverse-phase column (ACQUITY UPLC BEH C18, 1.7 μm, 2.1 × 150 mm; Waters) and a Xevo G2-XS QTof system (Waters) with a linear gradient from 1% B to 61% B. Buffer A: water with 0.1% (v/v) formic acid; buffer B: acetonitrile with 0.1% (v/v) formic acid.The percentages of remaining peptides were determined by using the peak area integration of the chromatograms.The obtained LC/MS data were analyzed using a MassLynx 4.1 (Waters).

RaPID selection of macrocyclic peptide binders to M pro
Taking advantage of tRNA GluE2 and tRNA Pro1E2 , we constructed a macrocyclic peptide library containing 3 kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β 1 ), (1S,2S)-2-aminocyclohexane carboxylic acid (β 2 ), and (1R,2R)-2-aminocyclopentane carboxylic acid (β 3 ) (Fig. 2).β 1 and β 2 were assigned to AUU and GCU codons using tRNA Pro1E2 GAU and tRNA Pro1E2 GGC , respectively.β 3 was introduced at the UGU codon using tRNA GluE2 GCA , because EF-P inhibits translation of β 3 when using tRNA Pro1E2 . 16For macrocyclization of the peptides, N-chloroacetyl-D-tyrosine ( ClAc y) and D-cysteine (c) were introduced at the initiator and elongator AUG codons using tRNA fMet CAU and tRNA Pro1E2 CAU , respectively.The thiol group of the c residue spontaneously reacts with the N-terminal chloroacetyl group of ClAc y to form a thioether bond for macrocyclization.Each npAA was precharged onto the respective tRNA using flexizymes.The peptide library comprised a repeat of 6 to 15 random residues encoded by using NNU codons (N = A, U, G, or C) flanked by the cyclizing ClAc y and c residues, followed by a GSGSGS linker connected to the 3′ end of the mRNA via a puromycin linker (Fig. 2c).The 3 cβAAs and the 12 pAAs (D, F, G, H, L, N, P, R, S, T,  Y, and V) were assigned to the NNU codons.
Table 1.Binding affinity, inhibitory activity, and serum stability of peptide inhibitors and their variants.The thioether bond is shown as a blue line.See Fig. 3 and SI Supplementary Fig. S3 for the structures of peptides.Sequences, read (%), kinetic association (k a ), dissociation (k d ), equilibrium (K D ), half-maximal inhibitory concentration (IC 50 ), and half-life in human serum (t 1/2 ) are shown.−: the kinetic values could not be accurately determined due to low affinity.
The macrocyclic peptide library was then applied to the RaPID selection against recombinant SARS-CoV-2 M pro .The random mRNA library was translated into the peptide library followed by conjugation of the peptide with the parent mRNA via a puromycin linker (SI Supplementary Fig. S1A).The mRNA/peptide conjugates were reverse transcribed into mRNA/cDNA/peptide complexes and applied to the affinity selection.The library was first subjected to naked magnetic beads to remove bead-binders and then applied to biotintagged M pro immobilized on streptavidin magnetic beads to recover M pro -binders.The bound fractions were recovered and amplified into the cDNA library by using PCR, followed by transcription into the mRNA library for the next selection round.By repeating the affinity selection, the recovery rate of M pro -binders increased at the third round, whereas that of bead-binders did not increase (SI Supplementary Fig. S1B).
The cDNA sequences and the corresponding peptides after the 3 rounds of selection were analyzed using next-generation sequencing (SI Supplementary Table S2 shows the top 100 sequences).Thirteen peptides containing β 1 or β 2 in their sequences were among the top 100 peptides, whereas β 3 was not found in the major family.Of the 13 peptides, we chose 4 peptides containing the yFHβ 1 motif at their N-termini (BM1, BM2, BM5, and BM6), 2 peptides containing a single β 1 and β 2 without the yFHβ 1 motif (BM4 and BM3, respectively), and 1 peptide containing 2 β 1 (BM7) for further analysis of their binding affinity, inhibitory activity, and serum stability (Table 1).BM1−7 were chemically synthesized using the standard solid-phase method without the C-terminal GSGSGS linker and their identities were confirmed by using MALDI-TOF MS (Fig. 3, SI Supplementary Figs.S2 and S3).

Biochemical activities and stabilities of peptide inhibitors
We first evaluated the binding affinities of BM1−7 to M pro by using SPR.BM2, BM3, BM5, BM6, and BM7 exhibited strong  binding affinities with K D values of 2.9 to 22 nM (Table 1 and SI Supplementary Fig. S4).Surprisingly, BM1 and BM4 showed substantially lower binding affinity with K D values of 1,300 and 1,200 nM, respectively, despite having high read frequencies.We next evaluated the inhibitory activity of BM1−7 against the hydrolytic activity of SARS-CoV-2 M pro using a reported MS-based method (Fig. 4a). 43BM2, BM3, and BM7, which have single-digit nanomolar K D values, showed particularly potent inhibition, with IC 50 values of 60, 40, and 20 nM, respectively.Peptides with weaker binding affinity exhibited IC 50 of 360 nM for BM5 and no inhibition for BM1 and BM4, implying a correlation between IC 50 and K D values.BM6 is an exception where the IC 50 value of 2,150 nM was not consistent with the K D value of 2.9 nM, likely due to its binding to a noncompetitive site of substrate.Notably, substituting the C-terminal tyrosine residue of BM2 for a phenylalanine completely abolished both binding and inhibition, highlighting the importance of single amino acid residues/specific functional groups for potent inhibition (Table 1); it is likely that the tyrosine hydroxyl group is involved in protein binding.
To evaluate contributions of the cβAA residues to potency, we synthesized variants of potent inhibitors with the cβAA being substituted for alanine (BM3A and BM7A, Table 1).Notably, the binding affinity and inhibitory activity of the alanine variants were completely lost, indicating that cβAA residues, namely β 2 in BM3 and β 1 in BM7, are essential for tight binding to M pro .
We also evaluated the half-life of potent cβAA-containing peptides and their variants in human serum, because the in vivo stability of peptide is one of the major issues in the development of peptide therapeutics. 46In general, peptides consisting only of L-α-amino acids are rapidly degraded in the range of minutes to a few hours by proteases in vivo. 47Each peptide and an uncleavable internal standard peptide were co-incubated in human serum at 37 °C; the relative amount of the remaining sample peptide was estimated by using LC/MS.The potent inhibitors BM2, BM3, and BM7, with IC 50 values of 20 to 60 nM, exhibited remarkably high peptidase resistance with half-lives (t 1/2 ) of more than 168 h (equivalent to a week), 48 h, and more than 168 h, respectively (Table 1, Fig. 4b, and SI Supplementary Fig. S5).By contrast, the alanine variants BM3A and BM7A showed substantially lower stabilities with half-lives of 4.6 and 4.2 h, respectively, highlighting the essential roles of cβAAs not only for efficient binding and inhibitory activity, but also for enhancing the proteolytic stability of peptides, probably because their folding ability stabilizes peptide structures.Our previous reports suggested that macrocyclic peptides are primarily digested by in-serum trypsin-like proteases at the C-terminal to Arg residues. 24,48BM7 has an Arg residue flanked by nonproteinogenic β 1 and c, which likely prevent the recognition of the Arg residue by proteases, resulting in its remarkably long serum half-life (more than 168 h).On the other hand, BM3 has a shorter half-life of 48 h compared with BM7, likely due to the protease susceptibility of the Arg residue distant away from β 2 by 4 amino acid residues.Unlike BM7 and BM3, BM2 exhibited a longer serum half-life (more than 168 h), whose resistance can be attributed to the absence of Arg in the sequence.

Conclusion
Our results highlight the significant contributions of β 1 and β 2 residues in the highly potent M pro inhibitory peptides BM7 and BM3 to their remarkable inhibitory activities (IC 50 of 20 and 40 nM, respectively) and serum stabilities (t 1/2 of more than 168 and 48 h, respectively).As shown in our previous crystallographic study of a FXIIa inhibitor, F3, in which 2 β 2 residues contributed to its inhibitory activity and stability via the formation of β-and γ-turn structures, 16 the strong folding abilities of β 1 and β 2 found in BM7 and BM3 are also expected to be crucial for their potency.The ribosomally mediated synthesis of libraries containing such nonstandard peptides, with multiple cβAAs and D-amino acids, was previously considered near impossible.However, the breakthrough development of tRNA Pro1E2 has enabled us to prepare a custom-made FIT system including EF-P that has allowed the preparation of such "foldamer-type" libraries, opening up an entirely new chemical space relative to that obtainable by using the natural transcription/translation machinery.
Compared with our macrocyclic peptides BM3 and BM7, other M pro inhibitory peptides reported to date exhibit limited activity and/or stability.A substrate-derived cyclic peptide inhibitor (UCI-1 49 ) and linear L-peptide inhibitors (p13 50 and compound 21 51 ) have been reported; however, their inhibitory activities are in the micromolar range.Eberle et al. designed all-D-peptide inhibitors based on the retro-inverso principle from phage display hit L-peptides, resulting in metabolically stable D-peptides with moderate inhibitory activity (IC 50 up to 1.57 μM). 52,53ohansen-Leete et al. screened potent thioether macrocyclic peptides from a ribosomally synthesized library consisting of standard pAAs in the random region and cyclizing ClAc y and L-Cys by means of the RaPID system (IC 50 up to 70 nM). 54Despite their high potency, stability against hydrolysis by M pro is limited, with 30% degradation after 1 h of incubation with 2.5 μM M pro , highlighting the importance of our results for the design of hydrolysis stable inhibitors of M pro and other disease-relevant enzymes.Xu et al. discovered potent disulfide macrocyclic L-peptides from a virtual screening (IC 50 up to 19 nM), although the lability of the disulfide bond under the physiological reducing conditions would eliminate their active forms and their serum stability has not yet been defined but is likely poor. 55he reported results indicate the difficulty of achieving high inhibitory activity against proteases and proteolytic resistance with peptides consisting only of α-amino acids.On the other hand, cβAAs are excellent building blocks that stabilize peptide structures with a minimal number of residues.In fact, although BM3 and BM7 have only 1 β 2 and 2 β 1 , respectively, they exhibited 2-digit nanomolar IC 50 values and remarkably long serum half-lives.We recently reported potent macrocyclic peptide M pro inhibitors containing cyclic γ 2,4 -amino acids (cγAA) that have rigid cyclic main chains. 24One resultant peptide, GM4, comprising 13 residues with a cγAA, exhibited an IC 50 value of 50 nM and a serum half-life of 126 h.A crystal structure of the M pro :GM4 complex revealed that the noncanonical cγAA residue binds to the catalytic subsite as a small amino acid surrogate and prevents hydrolysis by M pro .This observation underscores the potential of cγAAs as another promising building block of peptide therapeutics.Therefore, the combined use of cβAA and cγAA within a single peptide is a possible future strategy for discovering novel macrocyclic peptides characterized by both high potency and exceptional stability.These peptides hold great potential for diverse therapeutic applications, representing a captivating frontier in peptide-based drug development.

Fig. 2 .
Fig. 2. Incorporation of cyclic β-amino acids and D-amino acids into a peptide library by means of genetic code reprogramming.a) Structures of cβAAs used in this study.b) Reprogrammed codon table that consists of 3 cβAAs (β 1−3 ), 2 D-α-amino acids ( ClAc y and c), and pAAs.ClAc y was assigned at the initiator AUG codon and the others were assigned at the elongator codons.c) Sequence design of mRNA library and the corresponding peptide library.Peptides spontaneously macrocyclized between ClAc y and c via a thioether bond.The mRNA and peptide were covalently linked via a puromycin linker.

Fig. 1 .
Fig. 1.Secondary structures of tRNAs used for genetic code reprogramming.a) tRNA GluE2 .b) tRNA Pro1E2 .The T-stem motif for EF-Tu binding is in blue.The D-arm motif for EF-P binding is in green.

Fig. 4 .
Fig. 4. Inhibitory activity and serum stability of BM1−7 and their variants.a) Dose response analysis of peptides against M pro .The M pro inhibitory activities of peptides were investigated by using SPE purification coupled to MS analysis.Data are presented as mean values ± standard deviation, s.d.(n = 3−5).b) Serum stability assay of macrocyclic peptides.Each peptide and an internal standard peptide were co-incubated in human serum at 37 °C and the relative amount of each peptide to standard was estimated by using LC/MS analysis at each time point.Data are presented as mean values ± s.d.(n = 3).