Combining Chemical Protein Synthesis and Random Non-standard Peptides Integrated Discovery for Modulating Biological Processes

Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes Abhishek Saha, Hiroaki Suga, and Ashraf Brik* Cite This: Acc. Chem. Res. 2023, 56, 1953−1965 Read Online ACCESS Metrics & More Article Recommendations CONSPECTUS: Chemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications. Chemical protein synthesis allows a wide range of changes to the protein’s chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, D-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic D-peptide binders. Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates. Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field. ■KEY REFERENCES •Nawatha, M.; Rogers, J. M.; Bonn, S. M.; Livneh, I.; Lemma, B.; Mali, S. M.; Vamisetti, G. B.; Sun, H.; Bercovich, B.; Huang, Y.; Ciechanover, A.; Fushman, D.; Suga, H.; Brik, A. De Novo Macrocyclic Peptides That Specifically Modulate Lys48-Linked Ubiquitin Chains. Nat. Chem. 2019,11, 644−652. 1 This work describes the utility of chemically synthesized biotin-Lys48-linked diand tetra-Ub chains in the RaPID system for the selection of potent and selective macrocyclic peptide binders for these chains and the investigation of their cellular effects in biological processes. Received: March 23, 2023 Published: June 13, 2023 Articlepubs.acs.org/accounts © 2023 The Authors. Published by American Chemical Society 1953 https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 Downloaded via TECHNION-ISRAEL INST OF TECHNOLOGY on November 6, 2025 at 07:49:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. •Huang, Y.; Nawatha, M.; Livneh, I.; Rogers, J. M.; Sun, H.; Singh, S. K.; Ciechanover, A.; Brik, A.; Suga, H. Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-linked Diubiquitin by a Twofold Strategy. Chem.�Eur. J. 2020,26, 8022−8027. 2 This work describes ab initio RaPID selection and consequent rational mutagenesis for the selection process to improve the binding affinity of macrocyclic peptide toward Lys48-linked di-Ub. •Rogers, J. M.; Nawatha, M.; Lemma, B.; Vamisetti, G. B.; Livneh, I.; Barash, U.; Vlodavsky, I.; Ciechanover, A.; Fushman, D.; Suga, H. In Vivo Modulation of Ubiquitin Chains by N-Methylated Non-Proteinogenic Cyclic Peptides. RSC Chem. Biol. 2021,2, 513−522. 3 This work describes genetic code reprogramming with FIT to enable the construction of a highly nonproteinogenic cyclic peptide library for Lys48-linked Ub chains to obtain a drug-like peptide. •Vamisetti, G. B.; Saha, A.; Huang, Y. J.; Vanjari, R.; Mann, G.; Gutbrod, J.; Ayoub, N.; Suga, H.; Brik, A. Selective Macrocyclic Peptide Modulators of Lys63Linked Ubiquitin Chains Disrupt DNA Damage Repair. Nat. Commun. 2022,13, 6174. 4 This work describes the utility of chemically synthesized biotin-Lys63-linked diUb chains in the RaPID system for the selection of potent and selective macrocyclic peptide binders for these chains and an investigation of their cellular activity for the disruption of DNA damage repair. ■INTRODUCTION Proteins are a universally important class of macromolecules that perform countless vital functions in living systems. Most biological processes such as metabolism, signaling, and gene expression are mediated by antigen−antibody, enzyme− substrate, and DNA−protein interactions in which proteins are key partners in these processes. 5,6 In addition, the protein− protein interaction (PPI) is increasingly emerging as the core of many signaling pathways and serves as a tool to decipher the molecular basis of numerous diseases. 7 Therefore, academic and commercial drug discovery initiatives are now concentrating on a number of well-characterized PPIs that are connected to cell signaling and survival as therapeutic targets for cancer and other disorders. 8 The inhibition of PPI is an obvious way to influence specific biological processes in living systems, 7 yet inhibiting PPI is extremely challenging due to the large surfaces that are involved and the dynamic molecular changes, i.e., posttranslational modifications (PTMs), that occur to the proteins. Several strategies have emerged to achieve this goal such as fragment-based screening, protein engineering, high-throughput screening (HTS), virtual screening, phage display, covalent DNA display (CDT), and mRNA display. 9,10 Among all of Figure 1. Strategy for the discovery of macrocyclic peptide binders for POI using the combination of chemical protein synthesis and the RaPID system to modulate biological processes and possibly develop novel drugs. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1954 these, molecules that are selected by mRNA display are more specific and selective toward the target molecule, providing tremendous gravity in current drug discovery research. 10 A powerful approach to discovering such inhibitors is RaPID, which was reported by Suga and co-workers. 11 This enables the construction of massive libraries (>1012 unique sequences) of unique macrocyclic peptides composed of “natural productlike” building blocks by genetic code reprograming using the FIT system and screening for the discovery of de novo macrocyclic peptide ligands relevant in drug discovery. Most proteins produced today for biological and medical research come from living systems through recombinant expression. Notably, the chemical synthesis and semisynthesis of proteins are increasingly being used in these studies. Largely, total chemical synthesis of protein is helpful when the proteins to be made are harmful to the expression system or have other features such as PTMs and D-proteins that are difficult to program in the living organisms. 12−14 Combining the clear advantages of RaPID technology and synthetic protein chemistry provides a powerful approach to support basic research and generate novel chemical entities for drug discovery (Figure 1). This Account outlines an overview of the potential applications of the RaPID system utilizing synthetic proteins as the target for the selection of macrocyclic peptides for modulating specific biological processes through inhibiting PPI. ■CHEMICAL SYNTHESIS OF UNIQUELY MODIFIED PROTEINS Modern peptide synthesis enables the construction of peptides with unique reactivates that allow their chemoselective ligation employing different methods (e.g., native chemical ligation) to assemble long polypeptides that correspond to folded proteins. 15−17 In recent years, adopting flow technology in automated fast-flow peptide synthesizers also allowed rapid protein synthesis in a single shot from individual amino Figure 2. Chemical protein synthesis enables site-specific incorporation of unique elements into proteins, mainly PTMs, to generate uniquely modified proteins. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1955 acids. 18 These methods enable a great level of control of the protein composition with up to several hundred residues in length, 19−21 which is unattainable with ribosome-mediated biosynthesis. This, for example, allows chemists to precisely modify proteins with natural and unnatural modifications with a virtually unlimited number of functional groups in the protein’s sequence, such as PTMs and their mimics, fluorescent tags, reactive groups, affinity tags, and enrichment handles (Figure 2). 12,13 PTMs of different amino acid side residues significantly increase the chemical space accessible to Figure 3. RaPID system with in vitro reprogramed translation. (A) tRNA aminoacylation with an activated N-acyl-D-Trp or proteinogenic or nonproteinogenic amino acid(s) by a suitable flexizyme (ClAc group taken as a representative). (B) General scheme of the FIT system shows genetically programmed cyclization of a nonstandard peptide. (ClAc group taken as a representative). (C) RaPID selection of macrocyclic peptides is carried out in a single round of in vitro cyclic peptide selection. The FIT technique is used to translate an mRNA library that has been ligated with puromycin on its 3′end into a peptide-puromycin-mRNA conjugate. After exposure to the immobilized target of interest in the solid support, the pool is reverse transcribed to its complementary DNA (cDNA). Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1956 proteins, 22 which is constituted of the 20 canonical amino acids. The modified amino acid residues offer a means of controlling protein activity since they are frequently targeted by certain “reader” and “eraser” proteins. These changes have a significant impact on the proteome’s functional diversity and important protein characteristics including structure, activity, localization, solubility, and stability, which can then have an impact on how proteins interact with one another. 22,23 Therefore, harnessing the power of chemical protein synthesis offers great opportunities to prepare such uniquely modified proteins and use them in various studies. From a chemistry perspective, PTMs such as phosphorylation, methylation, acetylation, lipidation, and others can be generally and straightforwardly introduced to synthetic proteins (Figure 2). Such modified residues are most often brought in during solid-phase peptide synthesis (SPPS) from commercially available or readily accessible building blocks. On the other hand, the production of glycosylated or ubiquitinated proteins will require the adaptation of more challenging synthetic approaches that are reviewed in refs 24−27. The labeling strategies that result in the covalent attachment of different molecules to the target protein sequence significantly assist biochemical research. 28 In general, biotin is a useful label for protein immobilization of a solid support because of its extraordinarily strong ability to bind to avidin or streptavidin (Figure 2). Another unique example of the advantage of applying chemical protein synthesis is the ability to prepare proteins having inverse chirality, i.e., D-proteins. These unnatural proteins have been utilized in mirror image phage display for the discovery of D-peptide binders as well as in racemic protein crystallography. 29−33 Notably, typical peptide-based drugs’ long-standing problems, such as their short biological halflives and low cell permeability, can be addressed by macrocyclic D-peptide ligands produced through phase display selection. ■RAPID INTEGRATES THE FIT SYSTEM WITH A CONVENTIONAL MRNA DISPLAY Several advantages can be attributed to conventional mRNA display; however, the selection of the drug-like binder required advancement in the in vitro translation system. The easiest approach to reprogramming the mRNA-display for the selection of unnatural peptides is to replace natural amino acids from the PURE in vitro translation reaction with chemically identical unnatural analogs that are recognized by tRNA-synthetases. In this regard, Suga and co-workers developed the most effective approach, known as RaPID, which integrates the FIT system with the mRNA display. 11,34 This novel combination enables the rapid discovery of various de novo macrocyclic peptides with diverse unnatural motifs, such as cyclization and backbone methylation for the POI. The FIT system employs synthetic tRNA-aminoacylating ribozymes (flexizymes) to assist the synthesis of almost any aminoacyltRNA of interest that allows genetic code reprogramming with numerous nonproteinogenic amino acids (Figure 3A). Notably, the flexizymes cannot recognize the side chain of the activated amino acid and hence can serve as a generic tRNA-synthetase. The FIT system employs a relatively simple translation in which only the initiation codon is reprogrammed with an Nchloroacetyl-amino acid (N-ClAc-aa), which enables cyclization with Cys present at the C-terminus (Figure 3B). This was further upgraded with several other electrophilic centers such Figure 4. (A) Lys and Met positions at ubiquitin for chain extension. (B) Different forms of protein ubiquitination to form substratemonoubiquitinated, multiubiquitinated, and polyubiquitinated chains. Lys29-, Lys33-, Lys63-, and Met1-linked Ub chains form extended conformations while Lys6-, Lys11-, Lys27-, and Lys48-linked Ub chains form closed conformations. (C) Ub is activated by E1 activating enzymes, E2 conjugating, and E3 ligase enzymes for ubiquitination. DUBs can remove Ub moieties from polyUb chains to reverse the effects of substrate ubiquitination and recycle the Ub, or they can modify the length or type of linkage in the chain. Ub chains signal for different biological pathways, e.g., proteasomal degradation in the case of ubiquitinated proteins with Lys48-linked chains. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1957 Figure 5. Discovery of cyclic peptide modulator for Lys48-linked Ub chains. (A) Scheme of biotin-Lys48-linked di-Ub and biotin-Lys48-linked tetra-Ub synthesis. (B) RaPID selection for Lys48-linked tetra-Ub binding cyclic peptide, Ub4ix. (C) DUB inhibition assay: Lys48-linked tetra-Ub incubated with OTUB1 or USP2 in the presence of Lys48-linked Ub binding cyclic peptide Ub4ix. (D) α-Globin modified with hemagglutinin (HA)-labeled Lys48-linked tetra-Ub and 26S proteasome. In the presence of Lys48-linked Ub binding cyclic peptide Ub4ix or side a proteasome inhibitor MG132 (control), inhibited proteasomal degradation of α-globin. (E) Ub4ix stimulates earlyand late-stage apoptosis in HeLa cells, treated at 10 μM for 24 and 48 h. (F) Synthesis of cleavable and noncleavable dimers of Ub4ix cyclic peptide. Bars represent mean values. Adapted with permission from ref 1. Copyright 2019 Springer Nature. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1958 as 3-(chloromethyl)benzoic acid (mClBz) and 4- (chloromethyl)benzoic acid (pClBz). 11,35 The synthesis of noncanonical aa-tRNAs catalyzed by the flexizyme technology enables unnatural peptides composed of various nonproteinogenic building blocks such as N-acylated amino acids, D-amino acids, short peptides composed of different combinations of N-methyl-, D-, β-, or γ-amino acids, S-(ClAc-farnesyl)-D-Cys, N-methylchloroacetyl-amino acids, aryl acids, and alkyl acids. 11 A representative stepwise RaPID process shown in Figure 3C depicts genetic code reprogramming in FIT combined with the conventional mRNA display technology. The description of detailed procedures can be found in several other reviews. 11,36 Importantly, the RaPID platform is highly competent for selecting drug-like cyclic peptides, which was also discussed in other reviews. 11,37 ■EXAMPLES THAT COMBINE SYNTHETIC PROTEIN CHEMISTRY WITH THE RAPID PLATFORM The key advantages of chemical protein synthesis stem from the ability to generate proteins with particular PTMs and reflect their molecular composition in cells. This, in principle, should support us with unique targets for the RaPID selection method to push the selection system to another level and open critical doors for the development of unique modulators of a desired cellular function. Ubiquitination is the attachment of a Ub (76 amino acids) or polyUb chain to its substrate protein. 38 In this process, a cascade of E1, E2, and E3 enzymes collaborate to generate an isopeptide bond between the C-terminal Gly of Ub and the Lys side chain of diverse protein substrates. The consequent Ub addition to N-terminal methionine or any of the seven internal lysines (i.e., Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) of the preexisting Ub may generate polyUb chains with diverse linkages (Figure 4A,B). 39 On the other hand, deubiquitinating enzymes (DUBs) can dissemble such polyUb chains into mono-Ub. 40 Interestingly, mono or polyUb chain linkages on a substrate protein dictate the fate of the protein. For example, Lys48-linked polyubiquitination targets proteins for proteasomal degradation while other chain types take part in different signaling pathways, e.g., DNA damage and repair (Figure 4C). 41,42 Notably, a majority of drug targets concentrate on preventing the function of ubiquitinating E1-E3 and DUB enzymes or 26S proteosomes. 43 Unfortunately, targeting these enzymes has not yet furnished promising drug candidates, which encourages researchers to seek other potential targets. We believe that targeting Ub chains directly as the signaling code might be an actional avenue in modulating the Ub system and generating compounds with potential therapeutic applications. In this regard, we have been targeting various Ub chain linkages for the modulation of related biological processes by utilizing the combination of chemical protein synthesis and RaPID. 1−4,44 By utilizing the toolbox of chemical protein synthesis, Ub chains of different lengths and linkages with Figure 6. (A) The ab initio RaPID selection and consequent chemical mutation in its sequence to improve affinity of the parent peptide. (B) In vitro dose-dependent inhibition activity of mJ08-L8W against OTUB1 and USP2 enzymes. (C) The in vitro proteasomal inhibition activity of mJ08-L8W peptide was characterized on α-globin modified with Lys48-linked tetra-Ub. (D) mJ08-L8W stimulates early and late stage apoptosis in HeLa cells, treated at 10 μM for 24 and 48 h. (E) Dose-dependent apoptosis assay exhibited IC50 of mJ08-L8W 11.9 μM and 3.5 μM treated for 24 and 48 h, respectively. Bars represent mean values for all the cases. Adapted with permission from ref 2. Copyright 2020 John Wiley and Sons. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1959 biotin labels can be prepared with high homogeneity and workable quantities. In the following section, we highlight our recent studies in which we have employed the target Ub chain in the RaPID system to select the most effective cyclic peptide ligand(s) that bind with high affinity and specificity and show their biological activities. ■COMBINING RAPID AND CHEMICAL PROTEIN SYNTHESIS TO DISCOVER MACROCYCLIC PEPTIDE MODULATORS FOR LYS48-LINKED UB CHAINS De novo Macrocyclic Peptides That Inhibit Proteasomal Degradation and Induce Apoptotic Cell Death Combining the RaPID system and chemical synthesis of biotinylated Lys48-linked Ub, 45 we discovered de novo cyclic peptide Ub4ix that tightly binds to the Lys48-linked tetra-Ub (Figure 5A,B), with selectivity toward the chain length (mono-, di-, and tetra-Ub) and linkage type (Lys48 versus Lys63 or Lys11). The cyclic peptide Ub4ix was highly specific and tightly binds (KD= 6 ±1 nM) to Lys48-linked tetra-Ub yet exhibited weak affinity for the dimer (KD> 1 μM). Ub4ix also inhibited DUBs activity, in particular, OTUB1, which is selective toward the Lys48-linkage (Figure 5C). Our DUB inhibition assay and NMR study support the observation that the cyclic peptide Ub4ix primarily binds across the first three Ub units in Lys48-linked tetra-Ub. Moreover, the Ub4ix cyclic peptide inhibits 26S proteasomal activity in vitro by its strong attachment to Ub chains and prevents their identification and subsequent proteasomal processing (Figure 5D). When applied to cancer cells, Ub4ix can induce apoptosis in a manner similar to that of the strong proteasome inhibitor MG132 (Figure 5E). In addition, the binding affinity of the dimeric form of Ub4ix cyclic peptide to the Lys48-linked tetra-Ub and their cellular activity are interesting. 44 The dimer of Ub4ix with a cleavable disulfide bond, (Ub4ix_S7C)2, and with a stable thioetherbased linker, (Ub4ix_S7C)2CH2(Figure 7F), was found to bind tightly with the Lys48-linked tetra-Ub in vitro. Notably, these dimeric peptides are cell-permeable, particularly (Ub4ix_S7C)2CH2, which induces cell death in Hela cells and exhibits greater earlyand late-stage apoptosis in comparison to pG1 Ub4ix. Cyclic Peptide Modulators of Lys48-Linked di-Ub by ab initio RaPID Selection and Consequent Chemical Mutation for the Affinity Maturation Encouraged by the success of the first proof-of-concept for the modulation activity of Lys48-linked Ub chains by firstgeneration peptides (pG1), 1 we have devised ab initio RaPID selections 46 by using the previous peptide sequence library but cyclized by alternative thioether anchors to find next-generation macrocyclic peptides with improved affinity (Figure 6A). Notably, the use of mClBz and pClBz aromatic linkers instead of ClAc-amino acid for thioether bond formation in the macrocyclic peptide scaffold makes the peptides more rigid to produce a new library of macrocyclic peptides. 47 To achieve this, the translation at the starting codon (AUG) was engineered to encode mClBz or pClBz by the addition of flexizyme-precharged mClBz-tRNA or pClBz-tRNA to the FIT system. Only those cyclic peptides sharing the same sequence motifs with both mBz and pBz anchors exhibited a high recovery ratio over the background. The family of these peptides was referred to as the peptide generation 2 (pG2), and the best compound was found, mJ08,KD= 10 nm (Figure 7A), that improves the binding affinity by up to ∼10-fold compared to that of pG1 binders. Specific mutations to the common core motif of pG2 to mJ08 offer mJ08-L8W (KD= 1.2 nm) with an additional ∼8.5-fold binding affinity and high Figure 7. Discovery of drug-like cyclic peptides for the Lys48-linked tetra-Ub. (A) Distribution of amino acids including noncanonical amino acids in the trillions of cyclic peptides in the library targeting Lys48-linked tetra-Ub. In (B) HEK-293 and (C) U87 cells, cyclic peptide Ub4a induces improved apoptotic cell death over that of pG1 peptides (Ub4ix or mJ08-L8W) at 100 nM. (D) The luciferase-expressing human CAG myeloma cells in mice depict the tumor suppression activity of Ub4a in vivo similar to that of authorized anticancer drug bortezomib, and (E) the tumor suppression activity was quantified. Adapted with permission from ref 3. Copyright 2020 Royal Society of Chemistry. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1960 selectivity against other Ub probes such as Ub1, Lys11-linked di-Ub, and Lys63-linked di-Ub. The binding affinity and selectivity of mJ08-L8W, compared to those of pG1 compound Ub4i (KD= 90 nm), were supported by in vitro DUB inhibition assay, performed with Lys48-linkage-specific OTUB1 or the nonspecific USP2 (Figure 6B). The cellular inhibitory impact on the ubiquitinproteasome system (UPS) by mJ08-L8W was also investigated (Figure 6C). The involvement of UPS in the regulation of many of the apoptosis pathways 48 contended to test the effects of mJ08-L8W on the kinetics of apoptosis. mJ08-L8W evidently stimulated dose-dependent apoptosis to the 30− 70% level after 24 or 48 h of treatment on cancer cell lines (U87 and HeLa) selectively compared to normal cell line HEK293 (Figure 6D,E). Employing the ab initio RaPID selection strategy for cyclic peptide selection reveals beneficial mutations by using rational sequence alignment. This implies that minor modification in the anchor structure and peptide sequence may improve the parental macrocyclic peptide’s binding affinity. In vivo Modulation Activity of Lys48-Linked Ub Chains by Nonproteogenic Cyclic Peptides In general, cyclic peptides obtained by conventional RaPID selection were mostly proteinogenic and did not have drug-like properties, so the prospect of in vivo activity was uncertain. With the advantages of noncanonical amino acids in the sequence for drug development, 49−51 we applied the power of genetic code reprogramming in the RaPID process, 11 combining three N-methylated components (MeGly, MeAla, and MePhe), two D-stereochemistry components (D-Phe and D-Ala), and a linear hydrophobic side chain (Aoc) (Figure 7A). The selection process yielded a cyclic peptide Ub4a (MW: 1916 Da), which should have drug-like properties. 52 This peptide was found to be highly cell-permeable, block the action of DUBs and proteasome, induce apoptosis in cancer cell lines Figure 8. Discovery of a macrocyclic peptide modulator for Lys63-linked Ub chains. (A) Schematic synthetic strategy to obtain biotin-Lys63-linked Di-Ub, which was characterized by MS. (B) RaPID selection of the CP1 cyclic peptide that tightly binds to Lys63-linked di-Ub. (C) U2OS cell lysates blotted using γ-H2AX and H2AX antibody. Cells were treated with SCR (a CP2 scrambled sequence) and CP2. (D) γ-H2AX signal was quantified using Fiji (for n= 3 independent experiments). (E) Relative quantification of annexin-V positive cells after 96 h (n= 2). (F) Treated 293T cells were lysed and immunoprecipitated for flag and then blotted using flag and ubiquitin (Lys63-specific) antibody. (G) Lys63-linked Ub chains and their binders were pulled down with CP2 and SCR (using biotin-CP2 and biotin-SCR, respectively) and then blotted using antibodies for Lys63 and Lys48-linked Ub chains (n= 3). (H) Volcano plot showing the enrichment of protein levels involved in processes mediated by Lys63-linked Ub such as protein transport (blue), DNA repair (red), histone modification (green), and cell cycle (purple). A paired ttest was performed for z= 3 parallel samples. In all cases, values are the mean value and nrepresents the no. of biologically independent samples. Adapted with permission from ref 4. Copyright 2022 Springer Nature. Accounts of Chemical Research pubs.acs.org/accounts Article https://doi.org/10.1021/acs.accounts.3c00178 Acc. Chem. Res. 2023, 56, 1953−1965 1961