The regulation of cellular processes by ion channels has become central to the study of cancer mechanisms. Designing molecules that can modify ion channels specific to tumor cells is a promising area of targeted drug delivery and therapy. Despite their potential in drug discovery, venom peptides—a group of natural products—have largely remained understudied and under-characterized. In general, venom peptides display high specificity and selectivity for their target ion channels. Therefore, they may represent an effective strategy for selectively targeting the dysregulation of ion channels in tumor cells. This review examines existing venom peptide therapies for different cancer types and focuses on the application of snail venom peptides in hepatocellular carcinoma (HCC), the most common form of primary liver cancer worldwide. We provide insights into the mode of action of venom peptides that have been shown to target tumors. We also explore the benefit of using new computational methods like de novo protein structure prediction to screen venom peptides and identify potential druggable candidates. Finally, we summarize the role of cell culture, animal, and organoid models in developing effective therapies against HCC and highlight the need for creating models that represent the most disproportionately affected ethnicities in HCC.

The dysregulation of ion channels and receptors has recently emerged as an area of interest in the quest to find selective cancer therapies. Specifically, ion channels are involved in cancer cell migration, cell-cycle control, cell adhesion, and invasion and metastasis (1). For example, transient receptor potential (TRP) channels have proven to be important in the development of different malignant disease states. Calcium signaling which is indirectly regulated by TRP channels is involved in various cellular processes including cell death and apoptosis. When these processes are hijacked by tumor cells, the tumor gains capabilities such as drug resistance, migration, and proliferation. Breast cancer progression is reported to be supported by TRPV6 and more so by the gain-of-function R532Q mutant of TRPV6 (2). TRP channel overexpression has also been implicated in hematopoietic malignancies such as leukemia and lymphoma (3, 4). In colorectal cancers, TRP channels are reported to be overexpressed and responsible for facilitating metastasis (5). Overexpression of TRPV1 is reported to be associated with premalignancy preceding squamous cell carcinoma, and TRPM2 has been found to be upregulated in squamous cell cancer cell lines of the human tongue (5). High expression of different TRP channels have also been reported in liver tumors (5, 6). The pervasiveness of TRP channels has made them targets for dysregulation in cancer and TRP channels have been specifically targeted with therapies such as Englerin A, a tumor-selective treatment for renal cell carcinoma cell lines. Englerin A is an agonist of TRPC4 which induces cytosolic calcium upregulation, membrane depolarization and inhibition of growth (7).

Targeting overexpressed ion channels on the surface of tumor cells is a viable route to discovering druggable candidates with antitumor activity (8, 9). Venoms are composed of small molecules, peptides, and enzymatic proteins that, when purified, exhibit valuable pharmacologic activities (10, 11). Venom peptides are a class of compounds that can exert their pharmacologic effects by binding to ion channels or cell surface receptors with exquisite specificity. For this reason, venom peptides have been used as research tools to study biology of ion channels and receptors (12–14). Leveraging the selectivity of venom peptides to identify compounds that can manipulate dysregulated ion channels and receptors in tumor cells could produce cancer therapies with increased efficacy, while mitigating off-target effects and misfiring. Essentially, venom peptides would preferentially target tumor cells over healthy cells, leading to more effective cancer treatments that increase the quality of life for patients with cancer.

Venom peptides have been documented as potent antimicrobial and antitumor agents, chronic pain relief drugs, thrombin inhibitors, and antiplatelet drugs (Fig. 1) (10, 11). Therapeutics derived from venom peptides have provided novel paradigms for treatment, with marginal side effects. For example, the common side effects of captopril—the snake venom peptide—include rashes, proteinuria, loss of taste and paroxysmal cough, most of which are addressed in its derivatives enalapril and lisinopril and ramipril (15–17). In the case of the analgesic ziconotide, which targets N-type calcium channels, instead of opioid receptors that can lead to addiction, the side effects can include, constipation, diarrhea, nausea, dizziness, blurred vision, headache, vertigo, peripheral edema, ataxia, somnolence, dysarthria, and urinary retention (18). Eptifibatide, used to treat coronary syndrome, may result in bleeding and thrombocytopenia (19). However, the bleeding rapidly reverses, and thrombocytopenia is significantly less than the alternative therapy, abciximab. The most common side effect of exenatide, which is used to treat diabetes, is nausea (20). In most cases, when compared with alternative therapies, drugs derived from venom peptides possess reduced side effects.

Figure 1.

Therapies derived from venom peptides. Venom peptides from different organisms have been approved by international drug agencies and the FDA to treat diseases and disorders ranging from hypertension to pain. The only snail venom peptide derived drug currently in the market is ziconotide. This highlights the need for research of more snail venom peptides with therapeutic potential. Structures derived from the PDB, Universal Protein Resource (UNIPROT) and ChemDraw.

Figure 1.

Therapies derived from venom peptides. Venom peptides from different organisms have been approved by international drug agencies and the FDA to treat diseases and disorders ranging from hypertension to pain. The only snail venom peptide derived drug currently in the market is ziconotide. This highlights the need for research of more snail venom peptides with therapeutic potential. Structures derived from the PDB, Universal Protein Resource (UNIPROT) and ChemDraw.

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As with other natural products, the structure and function of venom peptides have served as scaffolds to drive the discovery of new bioactive compounds (21), and they continue to represent a major source of new medicines. Marine snails belonging to the Conoidea superfamily, which comprises Conidae (cone), Terebridae (terebrid), and Turridae (turrid) snails produce a significant amount of potentially pharmacologically relevant venom peptides. Over 800 species of cone snails have been estimated to produce over 100,000 different venom peptides called conotoxins (22). Of this number, 10,000 sequences have been deposited but only about 150 have been annotated and characterized (22, 23). The first conoidean drug is ziconotide (Prialt), which as mentioned before, is used to treat chronic pain in patients with HIV and cancer (24, 25). On the other hand, the venom peptides from Terebridae, teretoxins, although no less diverse, are significantly understudied, with only a few examples described in the literature (6, 26–32). Despite some similarities between conotoxin and teretoxin cysteine frameworks, teretoxins are not homologous to conotoxins and several findings suggest that they may function differently (26). Therefore, mining of the conotoxin and teretoxin chemical space will reveal additional peptides suitable for drug development. In this review, we will discuss recent insights into the use of venom peptides for cancer treatment, with a special focus on the application of snail venom peptides in hepatocellular carcinoma (HCC).

Global statistics and current treatment options for HCC

According to the World Health Organization, liver cancer was one of the most common causes of cancer death in 2020. The two most common forms of liver cancer are HCC and intrahepatic cholangiocarcinoma. HCC is the most common form of primary liver cancer worldwide accounting for approximately 90% of liver cancer related deaths. The incidence of HCC is estimated to reach greater than 1 million cases by 2025. HCC is unevenly distributed across different population as its highest incidence and death rate is seen in East Asia and Africa (33, 34). In the United States, HCC disproportionately affects African Americans, Asian, and Hispanics with African Americans experiencing the highest mortality rates due to HCC (35).

HCC has been reported to develop from a variety of risk factors such as infection with the hepatitis B virus, hepatitis C virus, alcoholism and cirrhosis, metabolic disorders such as nonalcoholic fatty liver disease, or nonalcoholic steatohepatitis (36). Due to this variation in the etiology of HCC, the staging of the disease considers both the extent of damage to the liver tissue and the tumor burden (37). Subsequently, choice of treatment considers the HCC stage as well as any associated liver disease, to maximize effectiveness while minimizing further liver damage and preserving liver function (36). Thus, early-stage HCC treatment commonly involves ablation and resection, with transplantation being considered for patients with larger tumor sizes. Patients diagnosed with early-stage HCC have a good prognosis and an overall survival of 5 years or more. Unfortunately, the survival statistics drop precipitously for patients diagnosed with intermediate and advanced stage HCC. Therapies for advanced HCC include multi-targeted kinase inhibitors sorafenib and lenvatinib (as first-line treatment), and regorafenib and cabozantinib (as second-line treatment). The second-line treatment also includes an antibody drug, ramucirumab. First-line therapies are estimated to provide a survival time of 11 to 13 months whereas second-line systemic therapies provide an estimated survival time of 8 to 10 months. More recently, additional antibody drugs, atezolizumab and bevacizumab, have been included as first-line treatment whereas sorafenib and lenvatinib are included as second-line treatment, and regorafenib, cabozantinib, and ramucirumab as third-line treatment. This change has improved the estimated survival for patients administered first-line treatment to over 19 months, 13 to 15 months after second-line treatment and 8 to 12 months following third-line treatment (33). However, despite continued progress, improvements have been slow and incremental, highlighting the need for different therapies with unique mechanisms of action.

Advances in gene sequencing technology, microscopy and similar techniques have significantly improved classification of HCC based on overexpression of certain markers enabling the development of targeted therapies against tyrosine kinases such as EGFR, RAF/RAS kinase, etc. In addition, the advent of immunotherapy options over the past few years has led to the recent FDA approval of several immune checkpoint inhibitors (atezolizumab, bevacizumab, nivolumab, and pembrolizumab), which have been reported to prolong survival after sorafenib failure, (38–40). A primary concern for these immunotherapy options in treatment of HCC is the variety of toxicities and immune-mediated adverse events that have been reported leading to discontinuation of treatment in some patients (36). There is an urgent and unmet need to identify druggable compounds in this space. Growing evidence suggests that venom peptides may represent an alternative therapeutic modality for treatment of HCC (6, 12, 41). In the following section, we summarize existing data on the performance of venom-derived peptides in various models of HCC.

Several HCC cell-lines and tissue express ion channels that are dysregulated (Table 1). In particular, HCC cell models are dysregulated in a range of ion channels, including potassium channels, sodium channels, aquaporin, and TRP channels. Potassium ion channels KCNQ1, KCNK2, KCNK15, and KCNK17 are reported to be down regulated in Hep3B, Huh7, and human HCC tissues samples whereas KCNK9, KCNJ11, KCa3.1, and KATP are overexpressed in human HCC tissues and cell lines (42–45). TRP channels are reported to be upregulated in both human and mouse HCC cell lines and tissues. TRPC6, TRPC1, TRPV2, TRPV4, TRPM7, and TRPV6 are upregulated in mouse and human HCC cell lines and tissues whereas TRPV1 is downregulated (6, 46–54). The prevalence of dysregulated TRP channels and potassium ion channels in HCC cell lines supports the emerging trend of targeting ion channels in anticancer drug discovery. Insights obtained in studies using venom peptides that are selective for these channels are likely to open new opportunities for cancer drug discovery or development of diagnostic tools (55).

Table 1.

Expression of various ion channels in HCC.

Sample typeChannelChannel full nameExpression levelRef.
Hep3B, HCC tissue KCa3.1 Calcium-activated potassium channel subfamily N member 4 Overexpression (42
Huh7, Hep3B KCNQ1 Voltage-gated potassium channel subfamily Q member 1 Downregulation (92
Human HCC tissue KCNK2 Two pore domain potassium ion channels Downregulation (43
 KCNK15 Two pore domain potassium ion channels Downregulation (43
 KCNK17 Two pore domain potassium ion channels Downregulation (43
 KCNK9 Two pore domain potassium ion channels Overexpression (43
Huh7, Hep3B KCNJ11 Inwardly rectifying potassium channel subfamily J member 1 Overexpression (44
HepG2, rat hepatocytes, Huh7 HFL KATP ATP sensitive potasssium channels Overexpression (45
HepG2, Huh7, whole mouse KCNH1 Voltage-gated potassium channel subfamily H member 1 Overexpression (93
HCC tissue TRPC6 Transient receptor potential channel canonical 6 Overexpression (46
Huh7 TRPC1 Transient receptor potential channel canonical 1 Overexpression (47,48
HepG2, and Huh7, DEN models TRPV1 Transient receptor potential channel vanilloid 1 Downregulation (49,50
HepG2, HepG2 xenografts, and Human HCC tissues TRPV2 Transient receptor potential channel vanilloid 2 Overexpression (51,52
HCC tissues TRPV4 Transient receptor potential channel vanilloid 4 Overexpression (53
HepG2 TRPM7 Transient receptor potential channel melastatin 7 Overexpression (94
1MEA TRPV6 Transient receptor potential channel Vanilloid 6 Overexpression (6
SNU449 T-type Ca2+ Voltage-gated calcium channels Overexpression (95
Huh7, Hep3B, SNU-387, PLC/PRF/5 P2×3 Purinergic receptor P2X ligand-gated ion channel 3 Overexpression (96
HCC tissues, MHCC97H CLIC1 Chloride intracellular channel 1 Overexpression (97–100
HepG2 VGSCβ1 Voltage-gated sodium channel beta subunit 1 Down-regulation (101
Rat HCC tissue Nav1.2 Voltage-gated sodium channel alpha subunit 2 Overexpression (102
Hep3B, Huh7 AQP5 Aquaporin 5 Overexpression (103
Huh-7, HLE, HepG2, SMMC-7721, HCC tissue AQP9 Aquaporin 9 Down-regulation (104
Sample typeChannelChannel full nameExpression levelRef.
Hep3B, HCC tissue KCa3.1 Calcium-activated potassium channel subfamily N member 4 Overexpression (42
Huh7, Hep3B KCNQ1 Voltage-gated potassium channel subfamily Q member 1 Downregulation (92
Human HCC tissue KCNK2 Two pore domain potassium ion channels Downregulation (43
 KCNK15 Two pore domain potassium ion channels Downregulation (43
 KCNK17 Two pore domain potassium ion channels Downregulation (43
 KCNK9 Two pore domain potassium ion channels Overexpression (43
Huh7, Hep3B KCNJ11 Inwardly rectifying potassium channel subfamily J member 1 Overexpression (44
HepG2, rat hepatocytes, Huh7 HFL KATP ATP sensitive potasssium channels Overexpression (45
HepG2, Huh7, whole mouse KCNH1 Voltage-gated potassium channel subfamily H member 1 Overexpression (93
HCC tissue TRPC6 Transient receptor potential channel canonical 6 Overexpression (46
Huh7 TRPC1 Transient receptor potential channel canonical 1 Overexpression (47,48
HepG2, and Huh7, DEN models TRPV1 Transient receptor potential channel vanilloid 1 Downregulation (49,50
HepG2, HepG2 xenografts, and Human HCC tissues TRPV2 Transient receptor potential channel vanilloid 2 Overexpression (51,52
HCC tissues TRPV4 Transient receptor potential channel vanilloid 4 Overexpression (53
HepG2 TRPM7 Transient receptor potential channel melastatin 7 Overexpression (94
1MEA TRPV6 Transient receptor potential channel Vanilloid 6 Overexpression (6
SNU449 T-type Ca2+ Voltage-gated calcium channels Overexpression (95
Huh7, Hep3B, SNU-387, PLC/PRF/5 P2×3 Purinergic receptor P2X ligand-gated ion channel 3 Overexpression (96
HCC tissues, MHCC97H CLIC1 Chloride intracellular channel 1 Overexpression (97–100
HepG2 VGSCβ1 Voltage-gated sodium channel beta subunit 1 Down-regulation (101
Rat HCC tissue Nav1.2 Voltage-gated sodium channel alpha subunit 2 Overexpression (102
Hep3B, Huh7 AQP5 Aquaporin 5 Overexpression (103
Huh-7, HLE, HepG2, SMMC-7721, HCC tissue AQP9 Aquaporin 9 Down-regulation (104

The Cancer Cell Line Encyclopedia available on EMBL-EBI's expression atlas provides valuable gene expression data showing differential expression of TRP channels in different liver cancer cell lines (refs. 56, 57; Supplementary Fig. S1). TRPC1 is overexpressed in almost all HCC cell lines followed by TRPV2 (measured in transcripts per million). This data provides strong support for targeting TRP channels overexpression in HCC.

Recent efforts in the field have identified several venom peptides that exhibit antitumor activity in HCC and other cancers (Table 2). Studies in the Holford lab identified terebrid snail venom peptide Tv1 exhibits selective antitumor activity in mouse HCC cell line 1MEA compared with nontumorigenic BNL CL.2 cell lines (6). Tv1 appears to inhibit calcium influx, which in turn results in downstream inhibition of cyclooxygenase-2 (COX-2) resulting in apoptosis (6). The anticancer activity of the bee venom peptide, melittin, has been extensively studied. The 26-amino acid peptide is reported to interact with the cell membrane resulting in membrane pore formation, loss of membrane function and cell lysis (58). Liver tumor metastasis in mice models was mitigated by administering alpha-melittin nanoparticles (59). The nanoparticles allow melittin to be administered intravenously thereby decreasing its toxicity to normal cells while improving toxicity against tumor cells. By targeting and regulating liver sinusoidal endothelial cells, alpha-melittin facilitates adaptive antitumor immune response in the liver thereby decreasing liver tumor metastasis and improving overall survival in mouse models (59). Melittin was also reported to reduce the viability of liver cancer cells, SMMC-7721 and Huh7 in a dose-dependent manner, reduce vasculogenic mimicry formation induced by CoCl2, and inhibit epithelial-to-mesenchymal transition (EMT) liver cancer cell lines and in vivo mouse models (60). Melittin prevents proliferation of HCC cell line, inhibit cell migration and tumor metastasis in vivo via RAC1 inhibition and inhibition of RAC1-dependent JAK activation (61). In HCC rats, crude viper venom improved liver enzyme levels and preserved hepatic architecture compared with untreated rats (62). Cinobufacini, an aqueous extract of the skin and venom gland of the Asiatic toad, induces apoptosis and decreases proliferation in HepG2 and BEL-7402 cell lines (63). This extract also contains bufothionine, resibufogenin, bufalin, and cinobufagin, compounds that are equally potent against HCC individually and act by inducing cell-cycle arrest and growth inhibition (64). Spider venom decreased proliferation, induced apoptosis, and decreased mitochondrial membrane potential in HepG2 cells (65). Low molecular weight peptides from the Iranian scorpion decrease the viability of 3D culture of HepG2 cells by inducing apoptosis (66). Snake venom and L-amino acid oxidase inhibit the proliferation of HepG2 cells and act synergistically with sorafenib to induce apoptosis (67). Batroxin, a peptide from Bothrops atox venom, decreases cell viability of HepG2 in an apoptosis dependent manner (68).

Table 2.

Overview of venom peptides reported to have anticancer activity and their mode of action.

TargetPeptideOrganismMode of action
Transient receptor potential ion channels Tv1 Terebra variegata Calcium dependent apoptosis (6
Cell membrane Melittin Apis mellifera Cell membrane disruption and lysis, Caspase C dependent apoptosis, inhibits metastasis (105
Vascular endothelial growth factor and fibroblast growth factor Hemilipin Hemiscorpus lepturus Inhibits angiogenesis and expression of proangiogenic factors (106
Chloride ion channels Chlorotoxin Leirus quiquestriatus Inhibits EMT (72,73
Cell membrane, cytoskeleton and extracellular matrix proteins Smp24 Scorpio maurus palmatus Cell membrane destruction, inhibition of migration via interaction with F-actin a, alteration of MMP-2/9 and TIMP-1/2 protein expressions (71
Sodium ion channels rAGAP Buthus martensii Cell-cycle arrest and apoptosis via upregulation of Bax and downregulation of BCl-2 (107
Cell membrane lysis Mastoparan Paravespula lewisii Mitochondrial-dependent apoptosis (108,109
Potassium ion channels KAaH1, KAaH2 Androctonus australis Inhibits migration, adhesion, and proliferation (110
Potassium ion channels Iberiotoxin Hottentotta tamulus Inhibits migration, adhesion, and proliferation (110
Calcium ion channels PnTx3-6, PhTx3-3 Phoneutria nigriventer Inhibits proliferation and cell viability (110
TargetPeptideOrganismMode of action
Transient receptor potential ion channels Tv1 Terebra variegata Calcium dependent apoptosis (6
Cell membrane Melittin Apis mellifera Cell membrane disruption and lysis, Caspase C dependent apoptosis, inhibits metastasis (105
Vascular endothelial growth factor and fibroblast growth factor Hemilipin Hemiscorpus lepturus Inhibits angiogenesis and expression of proangiogenic factors (106
Chloride ion channels Chlorotoxin Leirus quiquestriatus Inhibits EMT (72,73
Cell membrane, cytoskeleton and extracellular matrix proteins Smp24 Scorpio maurus palmatus Cell membrane destruction, inhibition of migration via interaction with F-actin a, alteration of MMP-2/9 and TIMP-1/2 protein expressions (71
Sodium ion channels rAGAP Buthus martensii Cell-cycle arrest and apoptosis via upregulation of Bax and downregulation of BCl-2 (107
Cell membrane lysis Mastoparan Paravespula lewisii Mitochondrial-dependent apoptosis (108,109
Potassium ion channels KAaH1, KAaH2 Androctonus australis Inhibits migration, adhesion, and proliferation (110
Potassium ion channels Iberiotoxin Hottentotta tamulus Inhibits migration, adhesion, and proliferation (110
Calcium ion channels PnTx3-6, PhTx3-3 Phoneutria nigriventer Inhibits proliferation and cell viability (110

Venom peptides can act as adjuvants or carriers for chemotherapeutic drugs, enhancing the specificity of these drugs. An example is the CPP-Ts peptide, a calcium ion modulator from the South American scorpion, Tityus serrulatus. CPP-Ts sub-peptide (aa 14–39) has been reported to show nuclear internalization specificity for cancer cell lines (HepG2, Caco-2, SK-MEL-188, MDA-MB-231, DU-145, A549) compared with normal immortalized counterparts (HUV-EC-C, MCR-5, HFF-1, HEK-293, BHK- 21, and MDCK; ref. 69). The mechanism of internalization is not fully understood, but CPP-Ts are being explored for use in chemotherapeutic drug delivery. The F7 venom fraction from the Moroccan cobra Naja haje has been reported to significantly decrease the viability of HCC multicellular tumor spheroids containing Huh7, WI38 human fibroblast cells, LX2 human hepatic stellate cell lines, and human endothelial cells [human umbilical vein endothelial cell (HUVEC)]. When compared with normal hepatocyte cells Fa2N-4 cells, the venom fraction showed selective toxicity for cancer cells in the MCTs. A mass spectrometric analysis of the F7 fraction revealed that it contained 36% neurotoxins, 1% cardiotoxins and 63% cytotoxins which enabled it to decrease cancer cell viability (70). These examples demonstrate the value of harnessing venom peptides in HCC anticancer research.

In a demonstration of the broad appeal of venom peptides for targeting tumor cells, Smp24, an antimicrobial peptide from the Egyptian scorpion, Scorpio maurus palmatus was reported to inhibit the proliferation and migration of A549 lung cancer cell lines by membrane disruption, dysregulation of MMP-2/-9 and TIMP-1/-2 and disruption of F-actin (71). Hemilipin, from Hemiscorpus leptirus, inhibits angiogenesis in HUVEC and CAM models, and chlorotoxin (CTX), also known as tumor paint, from Leiurus quinquestriatus (the death stalker scorpion) inhibits EMT in tumor allowing it to be used in outlines tumor cells in brain cancer (72, 73). These examples reiterate the value of harnessing venom peptides in anticancer research.

Collectively, the emerging evidence suggests that ion channels are dysregulated in cancers, and that venom peptides can be selective anticancer agents, including against difficult to treat cancers like HCC. In addition, these studies highlight that venom peptides exert their pharmacologic effects via a broad range of mechanisms, highlighting the unique potential of venom peptides for modifying cancer activity at various stages of tumor initiation and progression.

Insulin extracted from animal pancreas in 1922, was the first peptide drug (74). This breakthrough led to the search for other druggable peptides for treating diseases. Solid-phase peptide synthesis and high-performance liquid chromatography were major technological advances that decreased the turnaround time for peptide synthesis and drew attention to the field of developing peptide therapeutics (75). However, peptides have several important limitations that pose some challenges to their use as therapeutics. One of the major setbacks to drugging linear peptides is their short half-life and rapid clearance (76). In contrast, venom peptides circumvent this challenge as they are not linear, but rather fold into stable configurations through the formation of disulfide bonds that enable stability and decrease degradation from proteases. The rigidity of the disulfide bonds enables venom peptides to avoid the rapid enzymatic digestion fate of linear peptides. However, another limitation to investigating venom peptides as therapeutic leads is that the majority of venom peptides that have been sequenced have no solved structures. Currently, less than 2% of venom peptides have been structurally and functionally characterized. As mentioned, of 10,000 deposited conotoxin sequences (22, 77), only about 150 have been isolated and examined in more detail. There remains a large number of potentially potent and selective venom peptides to be identified and evaluated. The advent of new technologies that combine de novo and template-based structural modeling techniques with artificial intelligence enable the prediction of protein structures with a high degree of accuracy. In the last 3 years, deep learning approaches have made extraordinary advances in the accuracy of protein structure prediction. This was demonstrated by the performance of DeepMind's AlphaFold program (78, 79) at the 2018 and 2020 CASP meetings, which are blind tests of prediction algorithms as judged by independent assessors [including the Dunbrack group in 2004 (80) and 2014 (81)]. These approaches learn from the over 180,000 structures in the Protein Data Bank (PDB) and from amino acid sequence covariation through evolution. AlphaFold and RoseTTAFold have been reported to give a fairly accurate prediction of the structure of peptides and can provide a model with which interactions can be predicted for drug discovery (79, 82). AlphaFold2 has been shown to perform well both with and without template information (i.e., the structures of homologous proteins) and with only a small number of sequences in the multiple sequence alignment (∼30). It has also been shown to predict the structures of homo and heterooligomers. We used the ColabFold (83) implementation of AlphaFold2 to predict the structure of a panel of venom peptides including conotoxins, scorpion toxins, and teretoxins. The venom peptides displayed have either solution NMR or X-ray crystallography structures for all or a part of the sequences in the peptide. AlphaFold2 is able to produce a reliable prediction of the structure of these peptides without using templates (Fig. 2; ref. 79). Tv1 (the only teretoxin with a solved structure) was predicted without templates superimposed on its solved structure. The resulting model has an RMSD of 0.832Å indicating a high degree of similarity between the solved and predicted structures of TV1. Conotoxins and scorpion toxins with solved structures were also predicted with low RMSD values using AlphaFold2, indicating it can produce reliable predictions of the structures of venom peptides.

Figure 2.

New structure prediction tools show promise for predicting the structure of venom peptides without using experimentally solved structures as templates. A, RMSD values (in angstroms) indicate similarity between structures with values below 2.0 specifying high similarity between structures and vice versa. Some peptides like melittin have solution NMR and X-ray crystallography structures available for only a part of the sequence. i–iv, AlphaFold predicted structures (blue) versus solved structures (orange) for teretoxin Tv1, bee venom peptide – melittin, scorpion peptide—chlorotoxin, and wasp peptide—mastoparan. All structures in were predicted with AlphaFold2 using MMSeqs2 available online as ColabFold (82, 86). B, Cryo-EM structure of Omega conotoxin(ziconotide) bound to N-type calcium channel.

Figure 2.

New structure prediction tools show promise for predicting the structure of venom peptides without using experimentally solved structures as templates. A, RMSD values (in angstroms) indicate similarity between structures with values below 2.0 specifying high similarity between structures and vice versa. Some peptides like melittin have solution NMR and X-ray crystallography structures available for only a part of the sequence. i–iv, AlphaFold predicted structures (blue) versus solved structures (orange) for teretoxin Tv1, bee venom peptide – melittin, scorpion peptide—chlorotoxin, and wasp peptide—mastoparan. All structures in were predicted with AlphaFold2 using MMSeqs2 available online as ColabFold (82, 86). B, Cryo-EM structure of Omega conotoxin(ziconotide) bound to N-type calcium channel.

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With predicted structures available, it is possible to predict binding partners and interactions between venom peptides and their targets and this can facilitate rational drug screening and design. Docking tools such as HDock and ToxDock have also made it possible to predict the interaction between venom peptides and their targets as well as the residues essential for such interaction (84–86). Experimental structures of venom peptides and their targets can be used as training sets to improve docking models. Structure prediction tools like Alphafold2 when combined with docking methods like HDock or ToxDock, are valuable for identifying the peptides residues/domains important for binding to specific channels.

In addition to improving pharmacologic properties of venom peptides and our ability to predict and design their structure and function, developing improved models of HCC for testing the bioactivity of venom peptide is also important. Thus far, most pharmacologic assays have been done in two-dimensional (2D) HCC cell cultures and animal models of HCC. 2D cell cultures are easy to use and have facilitated biological research and discovery; however, these models fail to recapitulate the in vivo tumor microenvironment and model the interactions between different cell types required for tumor growth and migration. Furthermore, 2D cell culture models have altered phenotypic traits because the cell lines are immortalized to enable indefinite passages (87). Collectively, these factors affect our ability to confidently translate results from 2D cell line assays to clinical trials and underscore the need for models that more realistically represent tumor microenvironment and physiology.

Animal models can provide a more realistic representation of the tumor microenvironment. However, their use is limited due to cost, space, and maintenance requirements, as well as ethical concerns related to using animals in biomedical research. Recently, three-dimensional (3D) cell culture models, referred to as organoids, have emerged as a strategy for addressing these problems. Organoids can provide a more faithful representation of the 3D tumor microenvironment at a lower cost and with less maintenance than animal models. HCC derived from certain health conditions such as long-term infection with Hepatitis B virus can be propagated in organoids from healthy tissues allowing for flexibility in developing models from different disease states (88). Organoids also allow the development of functional organ-like structures from hepatocyte stem cells (89). Organoids from various tissues such as the stomach, kidney, cerebrum, and retina have been developed to understand diseases and biological mechanisms. However, human liver and liver cancer organoids have not been generated until recently (90). Broutier and colleagues showed the development of human primary liver cancer organoids which retained the characteristics of HCC, cholangiocarcinoma, and hepato-cholangiocarcinoma parent tissues from which they were taken after long-term expansion in culture (90). HCC organoids have also been reported to be developed from needle biopsies of patients; these organoids were reported to significantly recapitulate the genomic and histologic features of the parent tumor tissue in long-term culture, propagate in immunodeficient mice injected with the organoids and show sensitivity to sorafenib treatment (91). Collectively, these data indicate that organoids can provide a more holistic representation of the tumor microenvironment with moderate cost and effort. With these organoid models being developed, venom peptides with potential anti-HCC activity can be tested on a broader scale. Organoid technology is expected to play an increasingly important role in characterizing effects of venom peptides on HCC, as well as other malignancies.

Furthermore, we want to draw attention to a major issue that has important implications for discovery of HCC treatments for Black and historically underrepresented groups that are disproportionately affected by HCC. Although Black patients have lower survival rates compared with other groups, highlighting the urgent medical need, most current cell lines and models are not of Black origin. The only authenticated Black HCC cell line available commercially by the ATCC is Hep3B, a cell line obtained from an 8-year-old (Supplementary Table S1). The lack of relevant models creates a gap in basic research focused on understanding the molecular mechanisms and genetic differences governing the heterogeneity and tumorigenicity of HCC in these underserved populations. Therefore, deriving additional model systems from Black and other underrepresented patients should represent a priority for the field. Collectively, further improvements in model systems will facilitate and accelerate discovery and validation of pharmacologically valuable venom peptides and their derivatives.

In this review, we discussed the use of a class of under characterized and underutilized natural products, venom peptides, for modifying dysregulated ion channels and receptors in various cancers. We highlighted evidence that dysregulation of ion channels is associated with the predominant form of liver cancer, HCC, thus rationalizing the use of venom peptides as potential therapeutics in this disease area. Venom peptides have shown great promise in therapeutic development, although an approved venom cancer drug remains elusive. To further explore venom peptides and the opportunities that lie therein as anticancer therapies, it is essential that we employ a combination of computational strategies to elucidate venom peptide structures and develop effective biological models to test the efficacy of venom peptide candidates and leads. There is an urgent need to find new compounds for treating HCC, which affects thousands of patients and has low overall survival rates. The evolution of snails that eat fish, and scorpions that feed on rats illustrate the power of venom peptides in manipulating cellular physiologies. There is no question that these peptides could function to manipulate dysregulated ion channels in tumors. The big task is elucidating which peptides and what tumor channels, and this requires the advent of new models and new techniques.

Data Availability Statement

No new data were created or analyzed during this study. Data sharing is not applicable to this article.

No disclosures were reported.

The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NCI/NIH.

F. Achimba: Conceptualization, writing–original draft preparation, writing–review and editing, creation of figures and tables. B. Faezov: Writing–review and editing, creation of figures and tables. B. Cohen: Writing–review and editing, creation of figures and tables. R. Dunbrack: Writing–review and editing, creation of figures and tables. M. Holford: Conceptualization, writing–review and editing, supervision.

This review was supported by the JEDI award for article editing and Dr. Milka Kostic who provided guidance.

This project was (partially) supported by TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704 (5) from the NCI/NIH to M. Holford and to F. Achimba, NIH Pioneer Award 1DP1AT012812–01 to M. Holford, and the NIGMS R35 GM122517 award to R. Dunbrack.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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