Somatostatins are a family of natural cyclic peptide hormones expressed in pancreatic δ-cells, the gastrointestinal tract, neuronal cells, and certain tumors. Somatostatins can activate somatostatin receptors (SSTRs) which culminate in the inhibition of hormonal secretion, modulation of neuronal ion channel transmission, and cell growth arrest (59–61). SSTRs are a family of G-protein-coupled receptors (GPCRs), which include five subtypes termed SSTR1-5. The SSTRs incidence varies among different tumor types. Neuroendocrine tumors and tumors of nervous systems express high densities of SSTR2. But other tumor types, such as renal cell carcinoma (RCC), lymphoma, and inactive adenoma, have less SSTR2 or other SSTR subtypes (62, 63). The rationale for the oncology therapeutic use of SSAs depends on the expression of SSTR subtypes in relevant tumor tissues. The activation of SSTRs induced by SSAs results in the inhibition of tumor-associated pathophysiological hormonal secretion and tumor growth ( Figure 1A ) (61). Two macrocyclic peptide drugs derived from somatostatin, lanreotide ( Table 1 ; Figure 2A , compound 1) and pasireotide ( Table 1 ; Figure 2B , compound2), have been approved by the FDA in clinical practice up to now.

Lanreotide, the first-generation SSA, is a cyclic octapeptide. It has a high affinity to SSTR2 and less affinity to SSTR3 and SSTR5. Pasireotide, the second-generation SSA, is a cyclic hexapeptide. It has the same affinity to SSTR2 but a higher affinity to SSTR1, 3, and 5 than lanreotide (64, 65). Both have been approved by the FDA to treat Cushing’s disease and acromegaly, which are characterized by chronic hypersecretion of hormones from a pituitary adenoma ( Table 2 ) (14, 66, 67). In addition, lanreotide has been approved to treat neuroendocrine tumors. In addition, lanreotide has been approved to treat neuroendocrine tumors. It was reported that lanreotide inhibited the growth of the rat GH3 pituitary tumor cell line (IC₅₀ = 57 nM) (18). And, pasireotide inhibited the growth hormone release in rat pituitary cell lines (IC₅₀ = 0.4 ± 0.1 nM) (20). Multiple high-quality clinical trials and case series have demonstrated alleviated symptoms, a statistically significant increase in time to progression (TTP)/progression-free survival (PFS), long-term safety profile, and sustained antitumor effects upon treatment with lanreotide or pasireotide in patients with Cushing’s disease, acromegaly or neuroendocrine tumors (68–77).

On the other hand, SSAs may also have potential applications in non-endocrine tumor types because these tumors also express SSTRs. Thus, the off-label use of lanreotide or pasireotide to treat such diseases may also be promising. Despite the complete mechanisms of the antitumoral activity of SSAs not been demonstrated yet, some of the possible action mechanisms, including tyrosine kinase inhibition, induction of cell cycle arrest, proapoptotic effect, and inhibition of cancer cell adhesion and tumor angiogenesis, have been reported (78, 79). Lanreotide and pasireotide demonstrated antitumor effects in several preclinical models, including those for pancreatic ductal adenocarcinoma (PDAC), oral cavity squamous cell carcinoma, thyroid cancer, and prostate cancer (19, 21–26). However, the clinical data obtained from these non-endocrine tumors are still limited and discouraging. These two drugs showed no or limited benefit in several clinical cohorts (78, 80–83). The controversy between the preclinical and clinical data may result from uninvestigated SSTR statutes of enrolled patients and insufficient tumor cytotoxicity of SSAs. New generation SSAs with a better affinity to a broader range of SSTR subtypes and bigger tumor cytotoxicity and better planned clinical trials are needed to evaluate the role of SSAs in non-endocrine tumors.

CXCR4 is overexpressed in more than 20 human cancer types and correlated with advanced disease status and poor prognosis (84–86). The interaction between CXCR4 and its natural ligand CXCL12 (also known as stromal cell-derived factor-1α, SDF-1α) comprises a biological axis that is a well-validated PPI therapeutic target. It plays critical roles in mobilizing cancer cells and hematopoietic stem cells from the bone marrow to the peripheral blood and cancer progression, including the proliferation, invasion, and angiogenesis of cancer cells (87). Currently, the only marketed CXCR4 inhibitor is plerixafor (AMD 3100, a small-molecule drug) for stem cell mobilization in non-Hodgkin’s lymphoma (NHL) and multiple myeloma (MM) patients. Up to now, four macrocyclic peptide drugs serving as CXCR4 antagonists have been reported, and three of them are in clinical trials ( Table 2 ; Figure 1B ) (88, 89).

Motixafortide (BL-8040, BKT140) ( Table 2 ; Figure 3A , compound 3), a heterodetic cyclic peptide, is the first peptide antagonist for CXCR4 and receives orphan drug designation for the treatment of pancreatic cancer from the European Commission and the FDA. It was modified from a natural protein named T22 (27, 90, 91). The inhibition mechanism of motixafortide is different from that of plerixafor. While plerixafor works as a weak partial agonist, motixafortide works as an inverse agonist with a higher affinity and a more lasting CXCR4 occupancy, showing an IC₅₀ of 1 nM (28). It introduced unique cell-specific pro-apoptotic signaling pathways in MM and leukemia cells and induced apoptosis of acute myeloid leukemia (AML) blasts by altering the expression of miR-15a/16-1 (27, 29, 92). In addition, motixafortide may be an immune-modulatory agent by recruiting peripheral immune progenitor cells, which results in an enhanced antitumor immune response (93, 94). Both motixafortide monotherapy and combined therapy with chemotherapy or immunotherapy are safe with the profit from combination treatments in various cancer types (28, 30–34, 92). These cancers included AML, chronic myeloid leukemia (CML), MM, PDAC, and non-small cell lung cancer (NSCLC).

Balixafortide (POL6326) ( Table 2 ; Figure 3B , compound 4) is also modified from T22 by the protein epitope mimetic approach (95). The ADME properties of balixafortide demonstrated a higher binding affinity (100-fold) and a prolonged binding to CXCR4 than plerixafor (96). Binding to CXCR4 with the β-hairpin mimicry, balixafortide inhibited the CXCL12-induced activation of downstream MAPK-ERK/PI3K-AKT pathways in the lymphoma and AML cell lines and blocked the CXCL12-dependent chemotaxis in breast cancer and leukemia cell lines (35, 95, 97). In addition, balixafortide can enhance the efficacy of chemotherapy by mobilizing AML cells that locate in a protective stromal microenvironment into circulation and lead to prolonged survival in the murine leukemia model (98). The profiles with excellent safety and tolerability of this drug have been observed in three early-phase clinical trials (36, 99, 100). Additionally, balixafortide plus eribulin showed potential anti-cancer activities among patients with HER2-negative metastatic breast cancer in a phase I clinical trial (NCT01837095) (36). Further evaluation of the comparative efficacy and safety of this combination versus eribulin monotherapy among patients with metastatic breast cancer in a randomized phase III trial (NCT03786094) is ongoing.

LY2510924 ( Table 2 ; Figure 3C , compound 5) was developed by a medium throughput screen and rational design (37). It is a CXCR4 inhibitor featuring high in vivo stability but potentially with some safety issues and limited drug efficacy. Firstly, it showed reasonable preclinical activities in man tumor models. According to the structural modeling analysis, LY2510924 occupies the binding pocket in CXCR4 and possesses contacts with CXCR4 residues, including Arg30, Asp187, Arg188, Phe189, Gln200, His 113, Tyr190, and Glu288 (37). The potential mechanism of LY2510924 is that it may induce cell cycle arrest (G1 to G2-M progression) via inhibiting CXCL12-stimulated MAPK-ERK/PI3K-AKT and β-catenin pathways (37, 101). Monodrug therapy with LY2510924 demonstrated dose-dependent antitumor growth and anti-metastasis activities in leukemia, multiple solid tumor xenograft models, and a breast cancer metastatic model. When used in combination, it increased the efficiency of FLT3 inhibitors in preclinical FLT3-mutated AML models by suppressing TGF-β signaling (102). Secondly, the feature and advantage of LY2510924 is its high in vivo stability. The elimination half-life of LY2510924 is 9.16 hours (20 mg/day) in one phase I clinical trial, which was much higher than that of plerixafor (4.4-5.6 hours), motixafotide (0.29-0.72 hours) and balixafortide (5 hours) (28, 100, 103). Thus, a once-daily injection of LY2510924 for chronic treatment in clinical applications seems to be possible. However, the safety and clinical anti-cancer ability of LY2510924 need to be concerned. Serious adverse events occurred more frequently in the LY2510924 plus first-line standard of care (SOC) group than the SOC alone group (51% vs. 30.2%) in a small cell lung cancer (SCLC) phase II clinical trial (104). Additionally, the combination did not improve the drug efficacy over SOC monotherapy (median PFS: 5.88 months versus 5.85 months, p=0.98) (104). Similarly, LY2510924 also did not improve the drug efficacy when added to sunitinib in an RCC clinical trial (median PFS: 8.1 months versus 12.3 months) (105). Further investigation of LY2510924 to uncover the clinical mechanisms of LY2510924 is warranted.

Maro et al. (38, 106, 107) reported a novel macrocyclic peptide named Peptide R54 (Pep R54, Figure 3D , compound 6). According to the molecular dynamics results, the Arg4, 2-Nal5, and His6 side chains of Pep R54 occupied the minor and major pockets of CXCR4 (106). It mimiced CXCL12 to selectively bind the transmembrane bundle of CXCR4, which resulted in more efficient inhibition of CXCL12-mediated cell migration in a dose-dependent manner than plerixafor in vitro. In addition, Pep R54 displayed synergistic effects in combination with immunotherapy in a PES43 mouse xenograft model (38). These macrocyclic peptides of CXCR4 antagonists described above provide a valuable tool to target the CXCR4/CXCL12 axis in cancer therapy.

Over the past decades, inhibitory immune checkpoint blockade has become the fifth pillar of cancer treatment beyond surgery, chemotherapy, radiation, and targeted therapy. Inhibitory immune checkpoints are critical for maintaining self-tolerance and minimizing collateral tissue damage when responding to pathogenic infection. This complex immune response relies on an interplay of both adaptive and innate immune systems (108, 109). However, these checkpoints may be engaged in cancer development and progression, leading to tumor immune surveillance escape and suppressed antitumor immune responses (110). Thus, increasing efforts have been paid to target inhibitory immune checkpoints with ICIs to enhance anti-cancer immunity.

The PD-1/PD-L1 axis is a well-established T cell immune checkpoint that negatively controls the adaptive immune systems ( Figure 1C ). PD-1 is predominantly expressed on the surface of antigen-stimulated T cells. Upon binding to PD-L1, which is frequently expressed on cancer cells and antigen-presenting cells, the PD-1/PD-L1 interaction reduces T cell activation, proliferation, and survival leading to T cell exhaustion and protecting cancer cells from destruction mediated by cytolytic T cells (3, 4).

Several macrocyclic peptide inhibitors targeting the PD-1/PD-L1 axis have been developed. Bristol-Myers Squibb has reported two types of macrocyclic peptides for PD-1/PD-L1 blockade ( Figure 1C ) (111, 112). The first type of macrocyclic peptide includes BMSpep-57, BMSpep-71, and BMSpep-99 ( Figures 4A–C , compound 7, 8, and 9). The IC₅₀ values of these peptides were 9 nM, 7 nM, and 153 nM, respectively, in a homogeneous time-resolved fluorescence assay to determine their activities in inhibiting the interaction of PD-1/PD-L1 (39). These peptides restored the activities of the T-cell receptor-responsive promoter in vitro in a dose-dependent manner, and BMSpep-57 is the strongest one. The pharmacophore of these peptides is not related to other reported small-molecule PD-1/PD-L1 inhibitors, offering a blueprint for designing novel and more powerful antagonists of the PD-1/PD-L1 axis (113). The representative of another type of macrocyclic peptide developed by Bristol-Myers Squibb is BMS-986189 (compound 10), which completed the phase I clinical trial in 2018 (NCT0273973) in healthy people, but its structure was not released. BMS-986189 has a strong affinity for PD-L1 with an IC₅₀ value of 1.03 nM, according to the related patents (40). Gao et al. (41) also reported a macrocyclic peptide named C8 ( Figure 4D , compound 11) with high binding affinity with PD-1. The Arg5 and Cys9 of C8 form hydrogen bonds with the Thr76 and Asn 74 of the PD-1 to interfere with the PD-1/PD-L1 interaction. By interfering with the interaction and activating CD8⁺ T cells, C8 exerted antitumor effects in the CT26 mouse xenograft model in a CD8⁺ T cells-dependent manner.

On the other hand, due to the suboptimal response rates to ICIs targeting adaptive immune checkpoints, interest is growing in the innate immune checkpoints, especially the phagocytosis checkpoint (108). The signal-regulatory protein (SIRP)α-CD47 axis is the first identified tumor phagocytosis checkpoint. SIRPα, an inhibitory receptor, is expressed on myeloid cells such as macrophages and dendritic cells (DCs). It has an extracellular immunoglobulin (Ig)-like domain to bind its ligand CD47 which is often over-expressed on cancer cells. The interaction between CD47 and SIRPα transmits an inhibitory ‘do not eat me’ signal resulting in cancer cell evasion of immune detection and clearance (114–116). D4-2 is a macrocyclic peptide ( Figure 4E , compound 12) containing 15 amino acids. It was developed as an allosteric inhibitor to target the Ig-like domain of SIRPα ( Figure 1C ) (42). It can tightly bind to SIRPα and form multiple intramolecular hydrogen bonds and salt bridges. D4-2 can not only enhance the antibody-dependent cellular phagocytosis activity of macrophages for antibody-opsonized cancer cells in vitro but also demonstrate a synergistic effect on tumor growth or metastasis in combination with rituximab and TA-99 in vivo. Besides, the preclinical safety profile of D4-2 is also encouraging. Only weak declines in total cholesterol and blood urea nitrogen were observed in immunocompetent mice when treated with D4-2.

The HH signaling pathway plays a crucial role in embryonic patterning and development. However, the HH protein secreted by cancer cells induces the excessive activation of the HH signaling pathway in tumor-infiltrating stromal cells, which in turn contributes to the growth of cancer through several paracrine signals (117). Thus, the ligand-induced activation of the HH signaling pathway has emerged as one therapeutic target ( Figure 1D ). Owens et al. (43) identified a macrocyclic peptide named HL2-m5 ( Figure 5A , compound 13) based on the HH protein-binding loop of the HH-interaction protein ( Figure 1D ). The Trp4 and Met10 residues of HL2-m5 formed potential interactions with the HH protein. HL2-m5 inhibited ligand-dependent HH signaling pathway activation and suppressed HH signaling-dependent gene transcriptions in vitro. In addition, the inhibitory activity of HL2-m5 is superior to that of robotnikinin, which is a small-molecule inhibitor (IC₅₀: 250 nM vs. 15 μM).

RTKs are a family of transmembrane receptors that play pivotal roles in regulating cell-to-cell communications and various cellular processes. Abnormal activation of RTKs leads to many types of human malignancies. Thus, RTKs have become important targets for therapeutic intervention, and RTK-based cancer therapies have reached widespread clinical use nowadays (118, 119). Macrocyclic peptides have been proposed to interfere with RTK activation by disrupting the ligand-receptor PPIs.

Mesenchymal-epithelial transition (MET) tyrosine kinase receptor is encoded by MET proto-oncogene. The abnormal activation of MET by its natural ligand hepatocyte growth factor (HGF) triggers a multistep signal transduction cascade involved in tumorigenesis and seems invariably correlated with poor prognosis (120–122). Thus, the oncogenic role of HGF/MET signaling has underpinned the clinical investigation of MET inhibitors. HiP-8 ( Figure 1E , 5B , compound 14), a macrocyclic peptide consisting of 12 amino acids, is identified by Katsuya et al. (44). It allosterically inhibited the HGF/MET interaction by interacting with the NK4 and SP domains of HGF in a dose-dependent manner, which, in turn, prevented MET activation in vivo ( Figure 1E ). In addition, when labeled with ⁶⁴Cu, the HiP-8 variant served as a specific biomarker for noninvasive imaging of HGF-positive tumors using PET.

Other RTKs, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor (FGFR), are also frequently over-expressed in many forms of human malignancies. They play critical roles in the malignant growth and the progression of solid tumors (123, 124). Yin et al. (125), Imanishi et al. (126), and Stanton et al. (127) reported several macrocyclic peptides against EGFR and VEGFR by inhibiting the ligand-receptor PPIs. Similarly, Lipok et al. (128) reported a macrocyclic peptide, an FGF-FGFR interaction antagonist, which can block FGF-induced cell proliferation by 40%. Although more studies on the efficacy of these compounds are required, these macrocyclic peptides provide promising tools to target RTKs in cancer therapy.

Massive preclinical studies have suggested that the inhibition of autophagy may be a possible and powerful therapeutic strategy to improve outcomes in cancer patients (131, 132). Chloroquine and hydroxychloroquine are the only clinically available autophagy inhibitors (132, 133). However, the therapeutic windows of these two drugs are very narrow, and the toxicity caused by the drugs at therapeutically relevant doses illustrates the need to develop new drugs to target autophagy.

Gray et al. (45) identified a macrocyclic peptide named SUPR4B1w ( Figure 7 , compound 15), which targets microtubule-associate protein light chain (LC)3. LC3, as the core of the autophagy process, is essential for the maturation of the autophagosome (134). The Trp1 and Val5 residues of SUPR4B1w interacted with LC3 and further blocked autophagosome maturation in a dose-dependent manner and re-sensitized several resistant cell lines to cisplatin-mediated cytotoxicity ( Figure 6B ). Furthermore, the combination of SUPR4B1w with carboplatin induced almost complete inhibition of intraperitoneal tumor outgrowth in a mouse model of metastatic cancer.

The UPS plays important roles in many aspects of cell biological processes by marking proteins with ubiquitin followed by degradation of target proteins. The core of ubiquitination is the modification of protein substrates by ubiquitin (Ub) or polyUb chains. This process is precisely regulated by both the sequential interaction of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs) (135, 136). E3 ligases, as the key components at the last step of the ubiquitination cascade, are responsible for transferring ubiquitin to substrates and Ub chain topology. DUBs, conversely, cleave Ub from Ub chains to reverse the ubiquitination. Dysregulation of the UPS is associated with carcinogenesis, invasion, and cancer cell proliferation. Thus, targeting the UPS has been an attractive target for cancer therapy ( Figure 6C ) (137–139).

The Rat sarcoma (RAS) family of proto-oncogenes are the most frequently mutated oncogenes observed in about 30% of cancers. The protein members of this family include HRAS, NRAS, and KRAS. KRAS is the most commonly mutated isoform in cancers. When binding to GTP, small GTPases encoded by RAS are converted into active forms to stimulate downstream signaling pathways and regulate various cell functions. Mutated small GTPases encoded by oncogenic RAS are locked in an active state, thereby constitutively triggering downstream oncogenic pathways (153, 154). Due to a lack of suitable surface pockets for small-molecule inhibitors, RAS has once been considered an undruggable target (155). In 2021, based on several encouraging results from clinical trials, sotorasib, a small-molecule inhibitor, resulted in the FDA accelerated approval as the first KRAS-targeted therapy (156). In 2022, the FDA accepted the New Drug Application for adagrasib, another small-molecule inhibitor, for treating patients with NSCLC harboring the KRAS G12C mutation (157). Although progress has been made, targeting KRAS mutations remains a significant challenge in drug development.

Macrocyclic peptides against RAS have been shown as promising strategies to treat such RAS mutant cancers ( Figure 6D ). Buyanova et al. (158) reported a pan-RAS inhibitor named B4-27, a bicyclic peptide. It blocked the interaction between activated RAS and effector proteins, leading to the apoptosis of RAS-mutant cancer cells and suppressed tumor growth in vivo at low doses (≤5 mg/kg). However, pan-RAS inhibition may not be applicable in clinical because wild-type RAS is essential in normal cell signaling and may create toxicity concerns. In addition, different mutations in KRAS have distinct biochemical properties which may influence the therapeutic response (159). Thus, inhibitors against these specific mutants are being developed for RAS-targeted therapy.

KRpep-2d, a macrocyclic peptide, is the first reported selective KRAS-G12D inhibitor (160, 161). Although this compound has the disadvantages of low cellular activity and induction of mast cell degranulation, it provides a solid basis for the subsequent development of KRAS inhibitors (57). Based on its scaffold, two macrocyclic peptides, KS-58 ( Figure 9A , compound 22) and MP-3995 ( Figure 9B , compound 23) were developed with higher binding affinity, increased cell permeability, and better cellular activity. KS-58 is the first KRAS-G12D inhibitor with anti-cancer activity in vivo reported by Sakamoto et al. (56). KS-58 formed hydrophobic and cation–π stacking interactions with KRAS. In addition, KS-58 had synergistic growth inhibitory effects in the PANC-1 mouse xenograft model when combined with the drug gemcitabine. However, KS-58 required a high dose to show its efficacy despite no adverse side effects observe. Later, Lim et al. (57) reported another improved peptide named MP-3995 with anti-cancer activity in vitro. The favorable attribute is that MP-3995 only shows the antiproliferative effect in KRAS dependent cancer cell lines but has no effect in KRAS independent cell lines, displaying its high selectivity and potential lower toxicity. These compounds are still in their infancy but are likely to enter a clinical trial for KRAS mutant tumors. Additionally, Zhang et al. (162) reported three new macrocyclic peptides scaffold targeting the KRAS-G12D mutation, which was different from KRpep-2d. Cell-permeable macrocyclic peptides for KRAS-G12V mutation have also been developed by Pei et al. which blocked KRAS activated signaling pathway and induced apoptosis of cancer cells in vitro (163, 164). They may begin a new chapter in the discovery of KRAS inhibitors.