Bicyclic peptides, as the name suggests, are peptides containing two rings in their structure. Currently, there is no clear definition for the structural classification of bicyclic peptides. This article categorizes common types of bicyclic peptides based on their structures, specifically divided into the following four categories ( Figure 1).

Type I, two single-ring peptides connected. The linker can be a linear peptide chain or a small molecule linker, but the latter is more common, such as actinomycin D.

(2) Type II, where the bridging occurs within a macrocyclic peptide connected head-to-tail. This results in bicyclic peptides with shared segments between the two peptide rings; similarly, the bridging part can be a peptide chain or a small molecule rigid linker. Bicyclic peptides of Type II are predominant in nature, such as α-Amanitin and theonellamide F, and they can also be chemically synthesized through a two-step cyclization process.

(3) Type III, bicyclic peptides with a handle structure. Small molecules are used as the scaffold to connect the peptide chains, forming a structure similar to a "steering wheel". Various small molecules can be selected, and the construction of bicyclic peptides is relatively fast and convenient, making it more suitable for rapid large-scale synthesis. It is widely applied in the construction of bicyclic peptide libraries, especially those generated by biological methods.

Type IV, stapled peptides. Stapled peptides refer to linear peptides whose backbone has been modified by introducing cross-linkable groups, which are cyclized through methods such as olefin metathesis or click chemistry. Stapled peptides exhibit advantages including a high degree of α-helicity, enhanced cell membrane permeability, resistance to protease degradation, and a prolonged half-life in vivo^[12].

Structural modification of natural products as lead compounds is an important approach in the development of new drugs. Natural bicyclic peptides are widely distributed in nature and exhibit diverse biological activities, continuously inspiring the development of bicyclic peptide-based therapeutics.

Actinomycin D is an antitumor polypeptide antibiotic (Scheme 1a), a traditional drug for cancer treatment that was approved for tumor therapy in the 1960s^[13]. In 2016, Liu et al.^[14] combined Actinomycin D with RG1718 (an immunotoxin targeting mesothelin) and found that the two produced synergistic effects, enhancing antitumor activity in humans. Another naturally occurring bicyclic peptide already on the market is romidepsin, isolated from Chromobacterium violaceum found in Japanese soil samples. Romidepsin is a histone deacetylase inhibitor approved in 2009 for the treatment of cutaneous T-cell lymphoma. Its structure contains one natural amino acid, three non-natural amino acids, and one non-amino acid building block (Scheme 1b)^[15]. α-Amanitin is a highly toxic bicyclic octapeptide isolated from death cap mushrooms (Scheme 1c), which can selectively inhibit RNA polymerase II, producing toxicity toward eukaryotic cells and ultimately leading to apoptosis^[16]. In 2018, Matinkhoo et al.^[17] completed the total synthesis of α-amanitin, overcoming key challenges such as the enantioselective synthesis and diastereoselective sulfoxidation of the 6-hydroxytryptophan sulfoxide bridge, (2S, 3R, 4R)-4,5-dihydroxyisoleucine. α-Amanitin is now increasingly studied as an antibody-drug conjugate (ADC) in cancer therapy. For example, by conjugating an anti-EpCAM monoclonal antibody with α-amanitin, the resulting antibody-drug conjugate chiHEA125-Ama^[18] effectively eradicates experimental pancreatic cancer with low risk of systemic toxicity, potentially serving as a novel anticancer agent for pancreatic cancer and EpCAM-overexpressing tumors. In 2004, Potterat et al.^[19] isolated a new bicyclic 19-membered peptide, BI-32169 (Scheme 1d), from Streptomyces cultures, which showed strong inhibitory activity against human glucagon receptors. Kim et al.^[20] isolated six compounds from Bacillus sphaericus KCTC 12796BP cultures, including four bicyclic peptides (Scheme 1e) and two monocyclic peptides. Bioactivity testing revealed only the bicyclic peptides exhibited anti-allergic activity, while the monocyclic peptides were inactive, offering a new direction for developing anti-allergy drugs. Karim et al.^[21] discovered two bicyclic peptides, nyuzenamides A and B (Scheme 1f, Nyuzenamide A, B), from Streptomyces isolated from deep-sea sediments of the Sea of Japan; both compounds displayed antifungal activity and cytotoxicity. Subsequently, An et al.^[22] identified another bicyclic peptide, nyuzenamide C (1) (Scheme 1f), from the same source. Further bioactivity testing revealed that nyuzenamide C (1) exhibited antiangiogenic activity in human umbilical vein endothelial cells.

In addition to bicyclic peptides derived from microorganisms, many plants have also been found to contain bicyclic peptides. In 1989, a bicyclic peptide named theonellamide F^[23] (Scheme 2a), bridged by histidine and alanine residues, was reported. This peptide was isolated from secondary metabolites of marine plant sponges and exhibits antifungal activity and cytotoxicity. Subsequently, further separation and purification of the antifungal fraction from sponge extracts yielded five related peptides. Bioactivity assays demonstrated that all five peptides showed varying degrees of antifungal activity and cytotoxicity^[24]. Sunflower trypsin inhibitor-1 (SFTI-1) (Scheme 2b), extracted from sunflower seeds, is one of the smallest disulfide-bridged cyclic peptides discovered in nature^[25], as well as one of the smallest and most potent Bowman-Birk inhibitors (BBI)^[26]. Its structure is divided into two rings by a single disulfide bridge: one functional trypsin inhibitory ring (TI) and one non-functional secondary loop (SL), the latter of which can be replaced by bioactive peptides^[27]. SFTI-1 also exhibits antibacterial activity^[26] and pro-angiogenic activity^[27]. A bicyclic hexapeptide named RA-VII (Scheme 2c) was isolated from the roots of Rubia cordifolia L. and Rubia argyi (H. lsamv.). Studies have shown that this bicyclic peptide exerts potent antitumor activity by interacting with eukaryotic ribosomes and subsequently inhibiting protein synthesis^[28]. Celosia seed refers to the dried mature seeds of the plant Celosia argentea L., which belongs to the Amaranthaceae family. It is a commonly used traditional Chinese medicine for treating various eye diseases. A bicyclic octapeptide called moroidin^[29] (Scheme 2d) has been extracted from celosia seeds. Moroidin exhibits antitumor activity by inhibiting tubulin polymerization, with greater inhibitory activity on tubulin polymerization than colchicine^[30].

As previously mentioned, Sun et al. ^[33] constructed the first chemically synthesized small library containing 9 bicyclic peptides by using intramolecular thioester linkage on resin and dimedone-mediated disulfide formation off resin.

At present, the most commonly used method for constructing bicyclic peptide libraries via chemical approaches is the one-bead-one-compound (OBOC) high-throughput screening technique (Scheme 5). This technique was initially proposed by Lam et al. in 1992^[50], and involves performing a series of efficient chemical syntheses on microbeads, enabling each bead to display only one peptide. This allows rapid and large-scale synthesis and screening of peptides to obtain those with specific targets. Unlike biological libraries, OBOC libraries are more inclusive. They not only allow natural amino acids but also permit the inclusion of other compounds such as radioactive isotopes, fluorescent dyes, and D-amino acids within the library^[51]. In 2023, Yan et al.^[52] utilized the OBOC technique to develop an in vivo localization-targeted peptide library-on-bead (LIB) screening method, making in vivo screening using OBOC technology feasible and further expanding its range of applications.

However, after screening OBOC libraries, the structures of the hit bicyclic peptides cannot be determined. To address this limitation, Joo et al. reported a dual-compound method (OBTC). In the OBTC library, resin beads are separated into two distinct layers; the bead surface displays one cyclic peptide for screening against macromolecular receptors, while the interior contains a linear peptide with the same sequence serving as an encoding tag. During screening of the compound library, the target only binds to the outer bicyclic peptides, and the inner linear sequences do not cause interference. The selected bicyclic peptides can be sequenced via Edman degradation or mass spectrometry. In the construction of the OBTC library, bicyclic peptide cyclization is mediated by three amide bonds formed between a rigid small molecule scaffold, trimesic acid (TMA), an N-terminal amine, lysine protected with a monomethoxytrityl (Mmt) group, and the side chain of C-terminal L-2,3-diaminopropionic acid (Dap).

Lian et al.^[55] synthesized an OBTC library on ChemMatrix resin to screen for specific inhibitors targeting protein tyrosine phosphatase 1B (PTP1B), but the hit peptides obtained exhibited poor cell penetration. Subsequently, researchers fused cyclic peptides with cyclic cell-penetrating peptides to generate bicyclic peptides, which demonstrated improved cellular permeability while retaining the ability to recognize specific intracellular targets. This approach remained effective in testing cell-permeable inhibitors based on peptide-prolyl cis-trans isomerase (Pin1), indicating a certain degree of universality.

The main methods for the biosynthesis of bicyclic peptide libraries include phage display, ribosome display, and mRNA display. Phage display is the most commonly used method; Heinis et al.^[56] designed a peptide library composed of two sequences, containing six random amino acids and three cysteines, displayed on (non-disulfide) filamentous phage. The three cysteine residues were linked with the small molecule compound 1,3,5-tris(bromomethyl)benzene (TBMB) to construct bicyclic peptides (Scheme 6). The resulting chemical entities can be further modified and mutated selectively through enzymatic reactions (such as proteolysis). After multiple rounds of screening, a lead inhibitor (PK15) was successfully synthesized, capable of specifically targeting human plasma kallikrein and effectively blocking the intrinsic coagulation pathway in human plasma.

In addition to TBMB as a cyclic linker for peptides, Chen et al. ^[57] investigated the activity of the same peptide using different linkers (Scheme 7), and found that combining different linkers with random libraries might be a promising strategy for generating highly structurally diverse macrocyclic libraries based on the significant differences in obtained IC₅₀ values. The 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA), N,N',N''- (benzene-1,3,5-triyl)tris(2-bromoacetamide) (TBAB), and N,N',N''-(benzene-1,3,5-triyl)triacrylamide (TAAB) linkers developed in this report, along with TBMB, can be applied to phage peptide libraries containing >4×10⁹ different peptides to create >1.2×10¹⁰ peptide macrocycle libraries.

Sako et al.^[58] reported a novel peptide scaffold containing one Cys and three non-proteinogenic amino acids that selectively forms the desired crosslinks, suitable for the preparation of bicyclic peptide libraries with uniform backbones. Hacker et al.^[59] utilized the orthogonality between cysteine side chain alkylation with bis(bromoethyl)benzene and click chemistry to synthesize a series of cyclic or bridged bicyclic peptides. This strategy employs mRNA display technology to produce high-constraint bicyclic peptide libraries comprising >10¹³ precisely oriented topological structures. The bicyclization method is scaffold-free, thus allowing diverse ring attachment points and multiple bicyclic topologies.

SICLOPPS generates cyclic peptide libraries in cells by using protein trans-splicing. Its biggest advantage is the ability to easily, quickly, and effortlessly create libraries containing hundreds of millions of cyclic peptides. Moreover, this library can be combined with any cell-based assay to screen for members exhibiting desired phenotypes.^[60] Since STCLOPPS cyclic peptide libraries are generated within cells, functional analyses can be performed against various targets, enabling not only assessment of the affinity of each compound in the library but also its functionality toward specific targets.^[61]

Bicyclic peptide libraries have demonstrated significant application potential and research value. In both academia and industry, various bicyclic peptide libraries have been successfully applied to the screening of specific target molecules, the discovery of drug lead compounds, and the study of intermolecular interactions.

The method of forming conjugates between bicyclic peptides and drugs or toxins for targeted drug delivery has become a promising approach in oncology. The concept of bicyclic peptide conjugates originates from antibody-drug conjugates (ADCs), which have become a mainstream strategy for improving the pharmacodynamics of small molecule drugs. ADCs refer to highly potent small molecule therapeutic agents conjugated with monoclonal antibodies directed against specific targets, forming antibody-drug complexes^[62]. However, ADCs have several disadvantages, primarily including: 1) antibodies possess certain immunogenicity^[63], potentially causing immune responses such as allergic reactions, respiratory symptoms, and anaphylactic hypotension^[64], thus it is recommended to co-administer ADCs with antihistamines; 2) the large size of monoclonal antibodies (approximately 150 kDa) makes it difficult for ADCs to enter cells to exert their effects^[65], and it is estimated that only about 0.1% of the administered drug can reach the tumor tissue, representing the most critical drawback of ADCs; 3) ADCs have a long half-life in vivo^[66]. Although prolonged exposure may compensate for their poor tissue penetration, extended exposure also renders them susceptible to enzymatic degradation by circulating plasma proteases, gradually releasing the payload systemically^[67], thereby likely inducing systemic side effects; 4) antibodies are typically metabolized and eliminated via the liver, resulting in payload release in the liver and gastrointestinal tract, making hepatotoxicity and gastrointestinal toxicity common adverse effects associated with ADCs^[68]. The emergence of bicyclic peptide conjugates effectively addresses these drawbacks of ADCs. First, bicyclic peptides contain two peptide rings, both capable of target binding, offering higher affinity. Additionally, due to their much smaller molecular weight compared to antibodies, their tissue penetration capability is significantly enhanced, enabling rapid and effective delivery of drugs to tumor tissues. Second, bicyclic peptide ligands are easy to synthesize and conjugate, making the development process more time- and labor-efficient. Finally, the properties of peptides result in a shorter in vivo half-life and primarily renal elimination, limiting systemic exposure to the payload and minimizing damage to normal tissues caused by the conjugated drugs or toxins^[69]. Moreover, since the degradation products of bicyclic peptides are amino acids, they cause almost no toxic effects on the human body. Based on these advantages, we believe that bicyclic peptides hold great promise for application in the field of conjugate-based targeted therapy. Currently, bicyclic peptide conjugates have become a platform technology at Bicycle Therapeutics, used for selective delivery of chemotherapeutic agents or other therapeutics to disease sites. Bicyclic peptide conjugates can be further categorized into bicyclic drug conjugates (BDCs), bicyclic toxin conjugates (BTCs), and tumor-targeted immune cell agonists (TICAs), which differ essentially only in the type of payload conjugated, while sharing similar fundamental characteristics.

PPIs play important roles in various physiological processes, and selective modulation of PPIs has emerged as a novel therapeutic intervention strategy. Due to the large, flat, and featureless binding surfaces of PPI targets, small molecule compounds find it difficult to bind to them. Peptide drugs offer greater advantages in targeting PPIs, and bicyclic peptides have attracted significant interest from researchers because they exhibit higher affinity and specificity for PPIs compared to linear and monocyclic peptides.

Epidermal growth factor (EGF) has been shown to be involved in many types of solid tumors, including head and neck cancer, breast cancer, colon cancer, ovarian cancer, and non-small cell lung cancer^[80]. Therefore, the EGF-EGFR pathway has become a focus for cancer treatment. Although EGFR inhibitors are already available in clinical settings, most patients develop resistance, leading to reduced efficacy with long-term use. Moreover, drugs targeting EGFR are highly limited due to issues related to bioavailability and toxic side effects. In this context, Guardiola et al.^[81] reported that directly inhibiting EGF may produce better therapeutic outcomes, and peptide-based drugs can achieve this goal. Subsequently, this research group proposed the design of structure-based bicyclic constrained peptides and mimicked an interaction domain of EGFR^[82]. In addition to studying their interactions with EGF at the molecular level, the group also confirmed their potential to block the EGF-EGFR interaction in a specific receptor-ligand assay, and further demonstrated their therapeutic potential in human cancer cells overexpressing EGFR.

Screening PPIs from peptide libraries is a rapid and efficient approach. Lian et al.^[83] first selected planar structures as scaffolds to synthesize bicyclic peptide libraries. The planar scaffolds led the resulting bicyclic peptides toward an overall planar geometry, maximizing molecular surface area and thereby enabling interactions with flat protein surfaces. This type of bicyclic peptide may offer a general solution for inhibiting PPIs. Subsequently, this research group identified an effective antagonist, Anticachexin C1, through screening of a bicyclic peptide library targeting tumor necrosis factor alpha (TNFα), which inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced death.

Strategies to inhibit the interaction of p53 with murine double minute 2 (MDM2) and murine double minute X (MDMX) have proven to be a promising approach for cancer therapy^[84]. Utilizing bicyclic peptides to inhibit the PPIs between p53 and MDM2/MDMX offers greater advantages. Li et al.^[85] synthesized a bicyclic stapled peptide, p53-16, by combining an all-hydrocarbon stapling strategy with lactam bridges (Scheme 10), significantly improving its α-helicity and proteolytic stability. p53-16 exhibits nanomolar binding affinity toward MDM2 and MDMX, can penetrate cell membranes, and activates the p53 pathway to selectively inhibit tumor cell activity. RAS represents an attractive target for anticancer drugs; however, as a protein involved in intracellular PPIs, RAS has remained a highly challenging target. Trinh et al.^[86] screened a library of bicyclic peptides targeting the G12V mutant KRAS and optimized hits, yielding a moderately potent cell-permeable KRAS inhibitor capable of physically blocking RAS effector interactions and inducing apoptotic death of cancer cells. This compound may serve as a lead for further structural optimization, providing a powerful tool for the development of RAS inhibitors.

Peptide G1 (Scheme 11) is an 11-amino acid residue peptide macrocycle targeting the Src homology 2 (SH2) domain of growth factor receptor-bound protein 2 (Grb2). Quartararo et al.^[87] used G1 as a starting point to generate bicyclic peptides with higher affinity, selectivity, and resistance to degradation through a peptide stapling approach. After two rounds of iterative design, the bicyclic peptide BC1 was obtained (Scheme 11), exhibiting 60-fold greater potency and 200-fold higher selectivity than G1. Moreover, under conditions where G1 was completely degraded, BC1 remained fully intact after 24 h and showed less than 15% degradation after 48 h in buffered human serum. This indicates that BC1 possesses strong resistance to degradation and can achieve long-lasting effects in vivo. This peptide cyclization approach holds promise for the development of selective inhibitors targeting SH2 domains and other phosphotyrosine (pTyr) binding proteins, as well as numerous other PPI inhibitors.

Although many bicyclic peptides have shown good affinity and activity towards PPIs and can serve as lead compounds for further development, membrane permeability remains a challenging issue. Currently, common strategies to address this involve coupling bicyclic peptides with CPPs or incorporating cyclic CPPs, which are metabolically stable and significantly enhance cytoplasmic entry efficiency.

Bicyclic peptides have been found to be highly effective enzyme inhibitors/activators, particularly against extracellular enzymes, where the requirement for membrane permeability is low. In our discussion of natural bicyclic peptides, we mentioned many naturally occurring bicyclic peptides with enzyme inhibitory activity, such as α-Amanitin^[16], Romidepsin^[15], SFTI-1^[25], and others. In addition to natural bicyclic peptides, numerous synthetic bicyclic peptides also exhibit enzyme inhibition/activation activity.

First, it is worth mentioning Plecanatide, a guanylate cyclase agonist and one of the latest prosecretory compounds, which has been approved by the U.S. Food and Drug Administration for the treatment of adult chronic idiopathic constipation (Scheme 12)^[88]. After oral administration, Plecanatide exhibits biological activity only within the intestine without being systemically absorbed, thereby offering high safety and favorable therapeutic efficacy; it also represents one of the few currently available bicyclic peptide agonists.

Then, the main focus is on enzyme inhibitors. Plasma kallikrein (PKal), a member of the kinin–kallikrein system, is a serine protease that catalyzes the release of the bioactive peptide bradykinin, thereby causing inflammation, vasodilation, increased vascular permeability, and pain. PKal has been identified as a potential target for the treatment of diabetic macular edema (DME)^[89]. Using a technology combining phage display with chemical cyclization, Teufel et al.^[90] have identified highly selective bicyclic peptide inhibitors with nanomolar and picomolar potency (core structure shown in Scheme 13a) by incorporating unnatural amino acids and non-peptide bonds to enhance stability in biological matrices. This peptide inhibits bradykinin release in vitro and has demonstrated in vivo efficacy in a rat paw edema model and a diabetes-induced retinal permeability rodent model, making it a promising new therapeutic agent for diabetic retinopathy and diabetic macular edema. Recently, an effective and stable inhibitor was selected from a library of bicyclic peptide PKal inhibitor analogs. Named THR-149, preclinical experiments have shown that this bicyclic peptide can prevent diabetes-induced retinal leakage in diabetic rat models. Van et al.^[91] further demonstrated through a series of preclinical studies that repeated administration of THR-149 can reduce several key pathologies associated with DME in diabetic rats, such as retinal thickening and neuronal cell damage. These experiments confirmed that THR-149 is suitable for further clinical development and holds promise as a novel therapeutic agent for diabetes.

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease involved in the turnover of extracellular matrix (ECM) proteins and associated with tumor growth and invasion. It remains challenging to develop highly potent and specific small-molecule inhibitors targeting uPA, while macromolecular inhibitors such as monoclonal antibodies exhibit poor penetration into tumor cells. Therefore, peptide-based drugs can be prioritized for development. Angelini et al.^[92] isolated a bicyclic peptide inhibitor named UK18 (Scheme 13b) from a combinatorial peptide library, which exhibited a binding affinity toward human uPA that was 200-fold higher than that of the previously identified best monocylic peptide, upain-1. As a small, highly constrained bicyclic peptide (<2 kDa), UK18 possesses typical protein-like characteristics, including a large interaction interface with its target, along with favorable binding affinity and specificity. Harman et al.^[93] obtained a series of constrained bicyclic peptides by screening a highly diverse phage display library, which were further used to design bicyclic peptides with enhanced affinity and stronger inhibitory activity against angiotensin-converting enzyme 2 (ACE2). This novel class of bicyclic ACE2 inhibitors represents one of the most potent ACE2 inhibitors reported so far in vitro, and will serve as a powerful tool for further exploring ACE2 function and its potential therapeutic applications. Factor XII (FXII) inhibitors are important for studying proteases in the intrinsic coagulation pathway and suppressing contact activation during coagulation assays, and they also hold potential for antithrombotic therapy. However, synthetic FXII inhibitors developed to date have shown weak binding affinity and/or poor selectivity. Baeriswyl et al.^[94] generated and screened a new combinatorial library containing billions of structurally diverse bicyclic peptides to identify synthetic FXIIa inhibitors. The optimal bicyclic structure, FXII618 (Scheme 13c), showed an inhibition constant (K_i) of 22 nmol/L and exhibited over 2000-fold selectivity over other proteases. It effectively and selectively inhibited the initiation of the intrinsic coagulation pathway in both plasma and whole blood without affecting the extrinsic pathway. Protein tyrosine phosphatases (PTPs) mediate the execution and regulation of numerous cellular processes, such as signal transduction. Designing PTP inhibitors has long been a challenging goal due to the highly conserved positively charged active site structure shared among all PTPs, which requires negatively charged moieties for tight binding. However, negatively charged substances typically lack cell membrane permeability. Liao et al.^[95] directly screened a combinatorial library and developed a cell-penetrating bicyclic peptide-based inhibitor, PTP1B, specifically targeting T-cell PTP (TCPTP) (Scheme 13d). One of its rings contains a cell-penetrating motif (CPP), while the second ring harbors a target-binding sequence. This bicyclic peptide shows promise for development as a PTP inhibitor.

Some bicyclic peptides also exhibit receptor inhibitor activity; for example, BI-32169 mentioned previously¹⁹ (Scheme 1d) demonstrates strong inhibitory activity against the human glucagon receptor. Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor A165 (VEGF-A165) in endothelial cells. Jia et al.^[96] developed a specific peptide antagonist that blocks the binding of VEGF to NP-1. The bicyclic peptide EG3287 effectively inhibited the binding of VEGF-A165 to both porcine aortic endothelial cells expressing NP-1 and breast cancer cells expressing only the NP-1 receptor.

Dysregulation of Notch signaling is associated with several human diseases, including cancer^[97]. Therefore, the development of drugs targeting Notch holds great promise. Urech et al.^[98] utilized phage display technology to isolate from a combinatorial library a bicyclic peptide, FL-NRR17, which exhibits high-affinity binding to the NRR domain of the human Notch1 receptor (Scheme 14). Abnormal expression of the epidermal growth factor receptor Her2 is linked to various malignancies, including breast cancer. Diderich et al.^[99] screened a library of 66 bicyclic peptides and identified several bicyclic peptide ligands specifically targeting the epidermal growth factor receptor Her2. The best obtained bicyclic peptide binds to Her2 with a KD (equilibrium dissociation constant) value of 304 nmol/L. Unfortunately, the aforementioned bicyclic peptide ligands did not exhibit the desired activity in cell-based experiments. However, these peptide ligands could be used to modulate the conformation of other disease-related targets and hold potential applications in tumor imaging and therapy.

The issue of traditional antimicrobial drug resistance has severely hindered the progress of modern medicine. Both new antimicrobial agents and novel action targets represent key focal points for future antimicrobial drug development. As next-generation therapeutics for treating multidrug-resistant bacterial infections, antimicrobial peptides (AMPs) have their applications limited due to disadvantages such as susceptibility to proteolytic degradation and significant hemolytic activity^[100]. Strategies to enhance protease stability include substitution with D- or non-proteinogenic amino acids, N- or C-terminal acetylation or amidation modifications, pegylation, lipidation, and peptide cyclization^[101-102], among which peptide cyclization is the most widely applied strategy.

In 2019, Imai et al.^[103] identified a novel antibiotic named Darobactin, which possesses a unique bicyclic structure, by screening Photorhabdus isolates. This compound is highly effective against various Gram-negative pathogens and exhibits no activity against common commensal intestinal bacteria or human cell lines, indicating minimal impact on the human body and high safety. Adaligil et al.^[104] discovered peptide antibiotics composed of D-amino acids using the "mirror phage display technique"^[105]. Among them, the most active bicyclic peptide P14 (Scheme 15a) demonstrated good antibacterial activity against Staphylococcus aureus and MRSA, with MIC values of 8 ·g/mL and 32 ·g/mL, respectively. Although certain natural or designed antimicrobial peptides exhibit cytotoxicity toward mammalian cells, the bicyclic antimicrobial peptide P14 does not cause erythrocyte lysis nor show significant toxicity toward mammalian cells even at concentrations as high as 256 ·g/mL. These findings suggest that P14 could be further developed as a promising antimicrobial bicyclic peptide.

Cationic antimicrobial peptides (CAMPs) exert bactericidal activity through a membrane disruption mechanism, and bacteria must completely redesign the structure of their cell membranes to develop mutations, a process that requires a long time. Therefore, few bacterial strains have evolved resistance to CAMPs^[106]. He et al.^[107] synthesized four bicyclic peptides with different lysine sites using the cationic antimicrobial peptide OH-CM6 as a lead compound and 1,3,5-trimethylbenzene as a small-molecule scaffold to crosslink the ε-amino groups of three lysine residues. Among them, bicyclic peptide 6 (Scheme 15b) exhibited optimal serum stability and antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria, and multidrug-resistant bacteria. Additionally, bicyclic peptide 6 showed no hepatotoxicity or nephrotoxicity in vivo and holds promise as a novel therapeutic agent for treating multidrug-resistant bacterial infections.

In addition to the widely used screening of bicyclic peptide libraries, some new methods have been developed to obtain antimicrobial bicyclic peptides. For example, Di et al.^[108] applied the concept of chemical space, which is extensively utilized in small-molecule drug discovery, and employed a novel 2DP approach to calculate the chemical space (2DP can generate topological shapes and pharmacophore fingerprints of peptides), leading to the identification of antibacterial bridged bicyclic peptide 62b with activity against Gram-negative bacteria Pseudomonas aeruginosa (Scheme 15c). Compound 62b exhibits strong biofilm inhibition and dispersal activity and can enhance the effectiveness of polymyxins. The findings of this study are significant for addressing serious bacterial infections in hospital settings. Furthermore, the chemical space approach provides a general strategy for discovering bioactive peptides with unusual topologies, thereby expanding the structural diversity of peptide-based therapeutics.

Another direction in the development of antibacterial agents is to identify novel therapeutic targets. Sortase, a transpeptidase, represents a potential target for developing anti-infective drugs. Rentero et al.^[109] screened a large library of billions of bicyclic peptides targeting sortase and identified effective and selective inhibitors of sortase A (SrtA). Studies on the cocrystal structures of bicyclic peptides and their targets revealed that the shape of the bicyclic peptides perfectly complements the target, enabling high-affinity and highly selective binding to the target. The peptide 2 synthesized by this group (Scheme 15d) exhibited a K_i value of (2.4±0.3) μmol/L, significantly better than the best selective small-molecule SrtA inhibitors developed thus far.

Although antimicrobial peptides have improved resistance compared to traditional antibiotics, the development of bacterial resistance is inevitable. Continuous research and development of new antimicrobial compounds and discovery of novel antimicrobial mechanisms will help limit antibiotic resistance. At the same time, strictly restricting the use of antimicrobial agents in non-essential cases and appropriately combining different types of antimicrobial agents will further reduce the risk of drug-resistant bacteria.

Molecular imaging techniques play a crucial role in medical diagnostic research. Peptide molecules, after labeling, become a class of molecular probes that can be used for disease diagnosis, treatment, and post-treatment tracking. Bicyclic peptides are highly promising positron emission tomography (PET) tracers and other diagnostic imaging agents. Bicyclic peptides possess rapid tumor penetration characteristics, combined with high efficiency and chemical diversity, enabling excellent signal-to-background ratios and shorter delays between administration and imaging. Therefore, they provide high-contrast imaging probes for clinical diagnostics and demonstrate convincing additional potential in targeted therapy.

To detect early tumor cells, tumor-targeting peptides such as RGDs must be incorporated into imaging probes. Park et al.^[110] synthesized two new bicyclic RGD peptides by grafting aminocyclopentane (ACP) and aminocyclohexane (ACH) onto a tetrapeptide (RGDK) sequence, which were then conjugated with DOTA (Scheme 16a). These conjugates were radiolabeled with the radioactive metal ⁶⁴Cu and both showed high affinity for U87MG glioblastoma cells, superior to that of the monocyclic c(RGDyK), making them suitable as PET imaging agents. Fani et al.^[111] designed a bicyclic somatostatin analog, which was conjugated with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and labeled with ¹⁷⁷Lu and ⁶⁸Ga for in vitro internalization, efflux, pharmacokinetics, and imaging studies on tumor xenografts expressing somatostatin receptor subtypes 2 and 3. Biodistribution and PET/CT studies were performed on corresponding nude mouse models. The optimal conjugate, ⁶⁸Ga-AM3 (Scheme 16b), demonstrated clear tumor localization within 1 h after injection, visible kidneys, and negligible background, indicating its potential as an excellent imaging tracer, particularly for PET. Melemenidis et al.^[112] synthesized a cyclic RGD variant [c(RGDyK)] with high affinity for α_vβ₃, which was multivalently conjugated to iron oxide microparticles (MPIO). Under in vitro flow conditions, the binding properties of c(RGDyK)-MPIO to endothelial cells expressing α_vβ₃ were tested, followed by quantification of the contrast-enhancing effect of c(RGDyK)-MPIO in colon cancer and melanoma animal models, providing opportunities for early tumor detection and determining the suitability of specific therapies. Eder et al.^[113] identified a bicyclic peptide with subnanomolar affinity for MT1-MMP, a matrix metalloproteinase overexpressed in tumors. This peptide was conjugated via its N-terminus to the chelator DOTA, and after multiple experiments, the optimal imaging agent BCY-C2 (Scheme 16c) was obtained. In vivo studies showed high tumor uptake in mouse models.

For imaging-capable bicyclic peptide conjugates, tumor uptake can be further increased by enhancing the proteolytic stability of the peptides and prolonging the serum half-life through fatty acid conjugation. Certainly, tumor signals and in vivo selectivity can be further optimized by altering the position, composition, and size of the molecular spacer, as well as by using fatty acid derivatives with a certain affinity for albumin. These examples strongly demonstrate that bicyclic peptides have significant potential for application in imaging and contrast enhancement.