<i>In vivo</i> modulation of ubiquitin chains by <i>N</i>-methylated non-proteinogenic cyclic peptides

Rogers, Joseph M.; Nawatha, Mickal; Lemma, Betsegaw E.; Vamisetti, Ganga B.; Livneh, Ido; Barash, Uri; Vlodavsky, Israël; Ciechanover, Aaron; Fushman, David; Suga, Hiroaki; Brik, Ashraf

doi:10.1039/d0cb00179a

Cited by 18 publications

(21 citation statements)

References 55 publications

(74 reference statements)

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“…The mechanism by which the macrocyclic peptides described in this work enter the cells, and whether they permeate cells passively or actively, is as yet unknown. We have previously reported similar cases where the thioether-macrocyclic peptides did enter cells and actively inhibit enzyme or protein functions (Kawamura et al, 2017, Nawatha et al, 2019, Rogers et al, 2021.…”

Section: Discussionmentioning

confidence: 79%

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

Morgan

Ikenoue

Suga

et al. 2021

Preprint

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The SAGA complex is a transcriptional coactivator that plays multiple roles in activating transcription and is conserved from yeast to humans. One of SAGAs activities is the removal of ubiquitin from histone H2B-K120 by the deubiquitinating module (DUBm), a four-protein subcomplex containing the catalytic subunit, USP22, bound to three proteins that are required for catalytic activity and targeting to nucleosomes. Overexpression of USP22 is correlated with cancers with a poor prognosis that are resistant to available therapies. We used the RaPID (Random non-standard Peptides Integrated Discovery) system to identify cyclic peptides that are potent and highly specific inhibitors of USP22. Peptide binding did not impact the overall integrity of the DUBm complex as judged by small-angle x-ray scattering, indicating that the inhibitors do not disrupt subunit interactions required for USP22 activity. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. The macrocycle inhibitors we have identified in this work thus exhibit favorable drug-like properties and constitute, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module.

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Section: Discussionmentioning

confidence: 79%

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

Morgan

Ikenoue

Suga

et al. 2021

Preprint

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“…Novel cyclic peptide analogues with varied linkages may have an impact on the peptide’s physicochemical and pharmacological properties. 36 We wanted to apply our method to check the activity and permeability of known cyclic peptides that bind to Ub chains and modulate their properties. Therefore, we prepared the propargylated version of our previously reported peptide having the thioether linkage.…”

Section: Results and Discussionmentioning

confidence: 99%

Gold(I)-Mediated Rapid Cyclization of Propargylated Peptides via Imine Formation

et al. 2022

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In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30–60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov’s addition of the amine on the Au(I)-activated propargyl. This strategy was successfully applied to prepare efficiently (56–94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.

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“…The peptides successfully blocked the action of deubiquitinases by inducing apoptosis in a cellular assay and suppressed tumor growth in a mouse model. 39 In addition to the cases discussed above, the RaPID system has led to many teMPs against other target proteins as summarized in Figure 5, illustrating the versatility and power of this platform. As highlighted in these examples, the RaPIDderived teMPs have notable benefits in comparison with traditional molecular modalities.…”

Section: Potential Of the Thioether-closed Macrocyclic Peptides Obtai...mentioning

confidence: 92%

The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides

2021

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Conspectus Although macrocyclic peptides bearing exotic building blocks have proven their utility as pharmaceuticals, the sources of macrocyclic peptide drugs have been largely limited to mimetics of native peptides or natural product peptides. However, the recent emergence of technologies for discovering de novo bioactive peptides has led to their reconceptualization as a promising therapeutic modality. For the construction and screening of libraries of such macrocyclic peptides, our group has devised a platform to conduct affinity-based selection of massive libraries (>1012 unique sequences) of in vitro expressed macrocyclic peptides, which is referred to as the random nonstandard peptides integrated discovery (RaPID) system. The RaPID system integrates genetic code reprogramming using the FIT (flexible in vitro translation) system, which is largely facilitated by flexizymes (flexible tRNA-aminoacylating ribozymes), with mRNA display technology. We have demonstrated that the RaPID system enables rapid discovery of various de novo pseudo-natural peptide ligands for protein targets of interest. Many examples discussed in this Account prove that thioether-closed macrocyclic peptides (teMPs) obtained by the RaPID system generally exhibit remarkably high affinity and specificity, thereby potently inhibiting or activating a specific function(s) of the target. Moreover, such teMPs are used for a wide range of biochemical applications, for example, as crystallization chaperones for intractable transmembrane proteins and for in vivo recognition of specific cell types. Furthermore, recent studies demonstrate that some teMPs exhibit pharmacological activities in animal models and that even intracellular proteins can be inhibited by teMPs, illustrating the potential of this class of peptides as drug leads. Besides the ring-closing thioether linkage in the teMPs, genetic code reprogramming by the FIT system allows for incorporation of a variety of other exotic building blocks. For instance, diverse nonproteinogenic amino acids, hydroxy acids (ester linkage), amino carbothioic acid (thioamide linkage), and abiotic foldamer units have been successfully incorporated into ribosomally synthesized peptides. Despite such enormous successes in the conventional FIT system, multiple or consecutive incorporation of highly exotic amino acids, such as d- and β-amino acids, is yet challenging, and particularly the synthesis of peptides bearing non-carbonyl backbone structures remains a demanding task. To upgrade the RaPID system to the next generation, we have engaged in intensive manipulation of the FIT system to expand the structural diversity of peptides accessible by our in vitro biosynthesis strategy. Semilogical engineering of tRNA body sequences led to a new suppressor tRNA (tRNAPro1E2) capable of effectively recruiting translation factors, particularly EF-Tu and EF-P. The use of tRNAPro1E2 in the FIT system allows for not only single but also consecutive and multiple elongation of exotic amino acids, such as d-, β-, and γ-amino a...

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In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides

Abstract: Cyclic peptides containing unnatural amino acids can modulate Lys-48 ubiquitin chains in cells and animals.

Cited by 18 publications

References 55 publications

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

Gold(I)-Mediated Rapid Cyclization of Propargylated Peptides via Imine Formation

The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides

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