De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

Disulfide-Directed Multicyclic Peptide Libraries for the Discovery of Peptide Ligands and Drugs.

Multicyclic peptides with stable 3D structures are a kind of novel and promising peptide formats for drug design and discovery as they have the potential to combine the best characteristics of small molecules and proteins. However, the development of multicyclic peptides is largely limited to naturally occurring products. It remains a big challenge to develop multicyclic peptides with new structures and functions without recourse to the existing natural scaffolds. Here, we report a general and robust method relying on the utility of new disulfide-directing motifs for designing and discovering diverse multicyclic peptides with potent protein-binding capability. These peptides, referred to as disulfide-directed multicyclic peptides (DDMPs), are tolerant to extensive sequence manipulations and variations of disulfide-pairing frameworks, enabling the development of de novo DDMP libraries useful for ligand and drug discovery. This study opens a new avenue for creating a new generation of multicyclic peptides in sequence and structure space inaccessible by natural scaffolds, thus would greatly benefit the field of peptide drug discovery.

Design and Ribosomal Incorporation of Noncanonical Disulfide-Directing Motifs for the Development of Multicyclic Peptide Libraries.

The engineering of naturally occurring disulfide-rich peptides (DRPs) has been significantly hampered by the difficulty of manipulating disulfide pairing. New DRPs that take advantage of fold-directing motifs and noncanonical thiol-bearing amino acids are easy-to-fold with expected disulfide connectivities, representing a new class of scaffolds for the development of peptide ligands and therapeutics. However, the limited diversity of the scaffolds and particularly the use of noncanonical amino acids [e.g., penicillamine (Pen)] that are difficult to be translated by ribosomes greatly hamper the further development and application of these DRPs. Here, we designed and synthesized noncanonical bisthiol motifs bearing sterically obstructed thiol groups analogous to the Pen thiol to direct the folding of peptides into specific bicyclic and tricyclic structures. These bisthiol motifs can be ribosomally incorporated into peptides through a commercially available PURE system integrated with genetic code reprograming, which enables, for the first time, the in vitro expression of bicyclic peptides with two noncanonical and orthogonal disulfide bonds. We further constructed a bicyclic peptide library encoded by mRNA, with which new bicyclic peptide ligands with nanomolar affinity to proteins were successfully selected. Therefore, this study provides a new, general, and robust method for discovering de novo DRPs with new structures and functions not derived from natural peptides, which would greatly benefit the field of peptide drug discovery.

Redox-Regulated Conformational Change of Disulfide-Rich Assembling Peptides.

Disulfide bond formation is a common mechanism for regulating conformational changes in proteins during oxidative folding. Despite extensive studies of the use of multiple disulfide bonds to constrain peptide conformation, few studies have explored their usage in developing self-assembling peptides. Herein, we report that a thiol-rich peptide could fold into an amphiphilic ß-hairpin conformation through the formation of two hetero-disulfide bonds upon oxidation, subsequently self-assembling into a mechanically rigid hydrogel. Breaking disulfide bonds under reductive condition, the hydrogel exhibited a transition from hydrogel to solution. Molecular simulation revealed that intermolecular interaction between two tryptophan residues was indispensable for hydrogelation. This work is the first case of the use of multiple disulfide bonds to control conformational change and self-assembly, and provides a cell-compatible hydrogel material for potential biomedical application.

Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization.

N-terminal cysteine (Cys)-specific reactions have been exploited for protein and peptide modifications. However, existing reactions for N-terminal Cys suffer from low reaction rate, unavoidable side reactions, or poor stability for reagents or products. Herein we report a fast, efficient, and selective conjugation between 2-benzylacrylaldehyde (BAA) and 1,2-aminothiol, which involves multistep reactions including aldimine condensation, Michael addition, and reduction of imine by NaBH3CN. This conjugation proceeds with a rate constant of ∼2700 M-1 s-1 under neutral condition at room temperature to produce a pair of seven-membered ring diastereoisomers, which are stable under neutral and acidic conditions. This method enables the selective modifications of the N-terminal Cys residue without interference from the internal Cys and lysine residues, providing a useful alternative to existing approaches for site-specific peptide or protein modifications and synthesis of cyclic peptides.

Directed Disulfide Pairing and Folding of Peptides for the De Novo Development of Multicyclic Peptide Libraries.

Disulfide-rich peptides (DRPs) have been an emerging frontier for drug discovery. There have been two DRPs approved as drugs (i.e., Ziconotide and Linaclotide), and many others are undergoing preclinical studies or in clinical trials. All of these DRPs are of nature origin or derived from natural peptides. It is still a challenge to design new DRPs without recourse to natural scaffolds due to the difficulty in handling the disulfide pairing. Here we developed a simple and robust strategy for directing the disulfide pairing and folding of peptides with up to six cysteine residues. Our strategy exploits the dimeric pairing of CPPC (cysteine-proline-proline-cysteine) motifs for directing disulfide formation, and DRPs with different multicyclic topologies were designed and synthesized by regulating the patterns of CPPC motifs and cysteine residues in peptides. As neither sequence manipulations nor unnatural amino acids are involved, the designed DRPs can be used as templates for the de novo development of biosynthetic multicyclic peptide libraries, enabling selection of DRPs with new functions directly from fully randomized sequences. We believe that this work represents as an important step toward the discovery and design of new multicyclic peptide ligands and therapeutics with structures not derived from natural scaffolds.

Condensation of 2-((Alkylthio)(aryl)methylene)malononitrile with 1,2-Aminothiol as a Novel Bioorthogonal Reaction for Site-Specific Protein Modification and Peptide Cyclization.

Site-specific modification of peptides and proteins has wide applications in probing and perturbing biological systems. Herein we report that 1,2-aminothiol can react rapidly, specifically and efficiently with 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) under biocompatible conditions. This reaction undergoes a unique mechanism involving thiol-vinyl sulfide exchange, cyclization, and elimination of dicyanomethanide to form 2-aryl-4,5-dihydrothiazole (ADT) as a stable product. An 1,2-aminothiol functionality can be introduced into a peptide or a protein as an N-terminal cysteine or an unnatural amino acid. The bioorthogonality of this reaction was demonstrated by site-specific labeling of not only synthetic peptides and a purified recombinant protein but also proteins on mammalian cells and phages. Unlike other reagents in bioorthogonal reactions, the chemical and physical properties of TAMM can be easily tuned. TAMM can also be applied to generate phage-based ADT-cyclic peptide libraries without reducing phage infectivity. Using this approach, we identified ADT-cyclic peptides with high affinity to different protein targets, providing valuable tools for biological studies and potential therapeutics. Furthermore, the mild reaction conditions of TAMM condensation warrant its use with other bioorthogonal reactions to simultaneously achieve multiple site-specific modifications.

Biocompatible and Rapid Cyclization of Peptides with 2,4-Difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile for the Development of Cyclic Peptide Libraries.

We report a biocompatible and rapid reaction between cysteine thiols and 2,4-difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB), which enables the efficient cyclization of peptides in neutral aqueous solutions. The reaction was further applied to cyclize peptides displayed on the phage surface without reducing phage infectivity, thus affording high-quality cyclic peptide libraries useful for screening of cyclic peptide ligands. Using the DFB-cyclic peptide library, we identified ligands that can distinguish the pro-survival protein Bcl-xl from its close relative Bcl-2. Therefore, this study on one hand reports a useful reaction for the construction of cyclic peptide libraries, and on the other hand presents valuable hits for further design of selective Bcl-xl ligands.

Design and Synthesis of Disulfide-Rich Peptides with Orthogonal Disulfide Pairing Motifs.

Disulfide-rich peptides (DRPs) are a class of peptides that are constrained through two or more disulfide bonds. Though natural DRPs have been extensively exploited for developing protein binders or potential therapeutics, their synthesis and re-engineering to bind new targets are not straightforward due to difficulties in handling the disulfide pairing problem. Rationally designed DRPs with an intrinsically orthogonal disulfide pairing propensity provide an alternative to the natural scaffolds for developing functional DRPs. Herein we report the use of tandem CXPen/PenXC motifs ((C) cysteine; (Pen) penicillamine; (X) any residue) for directing the oxidative folding of peptides. Diverse tricyclic peptides were designed and synthesized by varying the pattern of C/Pen residues and incorporating a tandem CXPen/PenXC motif into peptides. The folding of these peptides was determined primarily by C/Pen patterns and tolerated to sequence manipulations. The applicability of the designed C/Pen-DRPs was demonstrated by designing protein binders using an epitope grafting strategy. This study thus demonstrates the potential of using orthogonal disulfide pairing to design DRP scaffolds with new structures and functions, which would greatly benefit the development of multicyclic peptide ligands and therapeutics.

A Doubly-Quenched Fluorescent Probe for Low-Background Detection of Mitochondrial H2O2.

Hydrogen peroxide (H2O2) is an important product of oxygen metabolism and plays a crucial role in regulating a variety of cellular functions. Fluorescent probes have made a great contribution to our understanding of the biological role of endogenous H2O2. However, fluorescent probes for H2O2 featuring aryl boronates can suffer from moderate turn-on fluorescence responses. Strategies that can reduce the background fluorescence of these boronate-masked probes would significantly improve the sensitivity of endogenous H2O2 detection. In this work, we propose a general and reliable double-quenching concept for the design of fluorescent probes with low background fluorescence. A new fluorescent probe was developed for the detection of endogenous H2O2 in mitochondria of live cancer cells. This probe exploits a boronate-driven lactam formation and an eliminable quenching moiety simultaneously (i.e., the double-quenching effect) to reduce the background fluorescence, which ultimately results in the achievement of a >50-fold fluorescence turn-on. A linear concentration range of response between 1 and 60 µM and a detection limit of 0.025 µM can be obtained. This study not only presents a highly sensitive fluorescent probe for the detection of H2O2 but also provides a new concept for the design of fluorescent probes with a previously unachievable fluorescence off-on response ratio for other types of ROS and many other biologically relevant analytes.

Ordered and Isomerically Stable Bicyclic Peptide Scaffolds Constrained through Cystine Bridges and Proline Turns.

Bicyclic peptides are attractive scaffolds for the design of potent protein binders and new therapeutics. However, peptide bicycles constrained through disulfide bonds are rarely stable or tolerant to sequence manipulation owing to disulfide isomerization, especially for peptides lacking a regular secondary structure. Herein, we report the discovery and identification of a class of bicyclic peptide scaffolds with ordered but irregular secondary structures. These peptides have a conserved cysteine/proline framework for directing the oxidative folding into a fused bicyclic structure that consists of four irregular turns and a 310 helix (characterized by NMR spectroscopy). This work shows that bicyclic peptides can be stabilized into ordered structures by manipulating both the disulfide bonds and proline-stabilized turns. In turn, this could inspire the design and engineering of multicyclic peptides with new structures and benefit the development of novel protein binders and therapeutics.

Design and Synthesis of Cross-Link-Dense Peptides by Manipulating Regioselective Bisthioether Cross-Linking and Orthogonal Disulfide Pairing.

Existing disulfide-rich peptides, both naturally occurring and de novo designed, only represent a tiny amount of the possible sequence space because natural evolution and de novo design only keep sequences that are structurally approachable by correct disulfide pairings. To bypass this limitation for designing new peptide scaffolds beyond the natural sequence space, we dedicate to developing novel disulfide-rich peptides with predefined disulfide pairing patterns irrelevant to primary sequences. However, most of these designed peptides still suffer from disulfide rearrangements to at least one to three possible isomers. Here, we report a general and reliable strategy for the design and synthesis of a range of structurally diverse cross-link-dense peptide (CDP) scaffolds with two orthogonal disulfide bonds and a bisthioether bridge that are not subject to disulfide isomerizations. Altering the pattern of cysteine and penicillamine generates hundreds of different CDP scaffolds tolerant to extensive sequence manipulations. This work thus provides many useful scaffolds for the design of functional molecules such as protein binders with improved proteolytic stability (e.g., designed by epitope grafting).

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation.

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully-unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Plasmon-Enhanced Ultrasensitive Surface Analysis Using Ag Nanoantenna.

Raman scattering and fluorescence spectroscopy permeate analytic science and are featured in the plasmon-enhanced spectroscopy (PES) family. However, the modest enhancement of plasmon-enhanced fluorescence (PEF) significantly limits the sensitivity in surface analysis and material characterization. Herein, we report a Ag nanoantenna platform, which simultaneously fulfills very strong emission (an optimum average enhancement of 105-fold) and an ultrafast emission rate (∼280-fold) in PES. For applications in surface science, this platform has been examined with a diverse array of fluorophores. Meanwhile, we utilized a finite-element method (FEM) and time-dependent density functional theory (TD-DFT) to comprehensively investigate the mechanism of largely enhanced radiative decay. PES with a shell-isolated Ag nanoantenna will open a wealth of advanced scenarios for ultrasensitive surface analysis.

Thioether-Bonded Fluorescent Probes for Deciphering Thiol-Mediated Exchange Reactions on the Cell Surface.

Study on the processes of the thiol-mediated disulfide exchange reactions on the cell surface is not only important to our understanding of extracellular natural bioreduction processes but to the development of novel strategies for the intracellular delivery of synthetic bioactive molecules. However, disulfide-bonded probes have their intrinsic inferiority in exploring the detailed exchange pathway because of the bidirectional reactivity of disulfide bonds toward reactive thiols. In this work, we developed thioether-bonded fluorescent probes that enable us to explore thiol-mediated thioether (and disulfide) exchange reactions on the cell surface through fluorescence recovery and/or cell imaging. We demonstrated that our thioether-bonded probes can be efficiently cleaved through thiol-thioether exchanges with exofacial protein thiols and/or glutathione (GSH) efflux. The exchanges mainly take place on the cell surface, and GSH efflux-mediated exchange reactions can take place without the requirement of pre-exchanges of the probes with cell surface-associated protein thiols. On the basis of our founder methodology, for the first time we demonstrated the interplay of exofacial protein thiols and GSH efflux on the cleavage of external thioether-bonded compounds. Moreover, given that an understanding of the process of GSH efflux and the mechanism on which it relies is crucial to our understanding of the cellular redox homeostasis and the mechanism of multidrug resistance, we expect that our thioether-bonded probes and strategies would greatly benefit the fundamental study of GSH efflux in living cells.

Biscysteine-Bearing Peptide Probes To Reveal Extracellular Thiol-Disulfide Exchange Reactions Promoting Cellular Uptake.

In recent years, delivery systems based on the incorporation of thiols/disulfides have been extensively explored to promote the intracellular delivery of biological cargoes. However, it remains unclear about the detailed processes of thiol-disulfide exchanges taking place on the cell surface and how the exchange reactions promote the cellular uptake of cargoes bearing thiols or disulfide bonds. In this work, we report the rational design of biscysteine motif-containing peptide probes with substantially different ring-closing property and how these peptide probes were employed to explore the thiol-disulfide exchanges on the cell surface. Our results show that extensive thiol-disulfide exchanges between peptides and exofacial protein thiols/disulfides are involved in the cellular uptake of these peptide probes, and importantly glutathione (GSH) exported from the cytosols participates extensively in the exchange reactions. Cysteine-glycine-cysteine (CGC)-containing peptide probes can be more efficiently taken up by cells compared to other probes, and we suggested that the driving force for the superior cellular uptake arises from very likely the unique propensity of the CGC motif in forming doubly bridged disulfide bonds with exofacial proteins. Our probe-based strategy provides firsthand information on the detailed processes of the exchange reactions, which would be of great benefit to the development of delivery systems based on the extracellular thiol-disulfide exchanges for intracellular delivery of biologics.

Proteolytic Unlocking of Ultrastable Twin-Acylhydrazone Linkers for Lysosomal Acid-Triggered Release of Anticancer Drugs.

Targeted prodrugs exploiting cleavable linkers capable of responding to endogenous stimuli have increasingly been explored for cancer therapy. Successful application of these prodrug designs relies on the manipulation of both stability and responsiveness of the cleavable linkers, which, however, are difficult to be finely regulated, particularly for acid-responsive acylhydrazone bonds. Here we developed a new class of peptide-bridged twin-acylhydrazone linkers (PTA linkers) displaying both an ultrahigh stability and a rapid responsiveness-highly stable in neutral and acidic conditions due to the effect of cooperativity between the two acylhydrazone bonds, easily cleavable in acidic conditions after enzymatically triggered unlocking of the two bonds. Moreover, our study shows the design of PTA-linked prodrugs and the proof-of-concept application of the PTA linkers for site-specific release of anticancer drugs into cancer cells.

Peptide Macrocycles Developed from Precisely Regulated Multiple Cyclization of Unprotected Peptides.

Peptide macrocycles have been attractive scaffolds for the development of ligands and inhibitors to proteins, which have the potential of being developed as potent drugs. Novel strategies for peptide macrocyclization should be of particular interest to peptide drug design and discovery. Herein, an efficient strategy for designing and synthesizing macrocyclic peptides, which relies on the precisely regulated and efficient one-pot cyclization of unprotected peptides with 2,3,5,6-tetrafluoroterephthalonitrile (4F-2CN), is reported. The peptide bicycles can be considered as novel structurally hyperconstrained peptide macrocycles constrained with a rigidifying ring-closing moiety, consisting of an N-terminal 6-membered ring and C-terminal 13-membered ring fused with the benzene ring of 4F-2CN. These novel macrocyclic peptide scaffolds would be intriguing and promising scaffolds for developing macrocyclic peptide inhibitors and targeting ligands for many proteins.

Tunable accessibility of dye-doped liposomes towards gold nanoparticles for fluorescence sensing of lipopolysaccharide.

Inspired by the primitive role of lipopolysaccharide (LPS) and taking advantage of the membrane-philic properties of amphiphilic gold nanoparticles (AuNPs), we established a facile and efficient fluorescence turn-on detection strategy for LPS. Upon binding onto the surface of liposomes, LPS can tailor the accessibility of liposomes towards AuNPs, reminiscent of its primitive function on the surface of bacteria. Thus, while the fluorescence of the dyes labeled on liposomes can be markedly quenched by the membrane-philic AuNPs, the quenching effect can be efficiently prevented by the surface-bound LPS. The de-quenching effect is highly selective to LPS, relative to other negatively charged bio-analytes, which is due to not only the extremely high affinity of LPS to lipid bilayers, but also the unique molecular structure of LPS. Furthermore, this easy-to-construct method offers a limit of detection of ∼0.65 nM, which is comparable to that obtained from the superb synthetic sensors for LPS reported in the literature. This study would open up a new route for the design of sensing systems for LPS exploiting its unique structural pattern and primitive function.

A lysosome-locating and acidic pH-activatable fluorescent probe for visualizing endogenous H2O2 in lysosomes.

There is increasing evidence indicating that lysosomal H2O2 is closely related to autophagy and apoptotic pathways under both physiological and pathological conditions. Therefore, fluorescent probes that can be exploited to visualize H2O2 in lysosomes are potential tools for exploring diverse roles of H2O2 in cells. However, functional exploration of lysosomal H2O2 is limited by the lack of fluorescent probes capable of compatibly sensing H2O2 under weak acidic conditions (pH = 4.5) of lysosomes. Lower spatial resolution of the fluorescent visualization of lysosomal H2O2 might be caused by the interference of signals from cytosolic and mitochondrial H2O2, as well as the non-specific distribution of the probes in cells. In this work, we developed a lysosome-locating and acidic-pH-activatable fluorescent probe for the detection and visualization of H2O2 in lysosomes, which consists of a H2O2-responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore. The response of the fluorescent probe to H2O2 is significantly more pronounced under acidic pH conditions than that under neutral pH conditions. Notably, the present probe enables the fluorescence sensing of endogenous lysosomal H2O2 in living cells without external stimulations, with signal interference from the cytoplasm and other intracellular organelles being negligible.