Open access peer-reviewed chapter
Abstract
Pathogenic viruses causing large outbreaks often contain Class I fusion proteins on their envelopes. These proteins facilitate virus-host cell membrane fusion through a common mechanism involving the N-terminal Heptad Repeat region forming a coiled-coil trimer and the C-terminal region folding back to form a six-helix bundle (6-HB). Fusion inhibitors, particularly peptide-based ones like Enfuvirtide and Albuvirtide, target the 6-HB to block membrane fusion and have been clinically approved. Researchers have always been committed to digging out more peptide-based fusion inhibitors lead candidates and better molecular mechanisms. More compound structural optimization may predominate in future research, which we believe will lay a solid foundation for improving drug performance. This chapter offers a comprehensive review of the structure and function of 6-HB, along with the latest developments in peptide-based fusion inhibitors. Our insights aim to serve as a handy guide for researchers, offering inspiration and valuable information in this field.
Keywords
- HIV-1
- antivirals
- 6-HB
- fusion inhibitors
- peptide
1. Introduction
Both human health and the advancement of social civilization face considerable harm from emerging viral infectious diseases. For example, Acquired Immune Deficiency Syndrome (AIDS) is a devastating infectious disease caused by the Human Immunodeficiency Virus type 1 (HIV-1). The latest Joint United Nations Programme on HIV/AIDS (UNAIDS) reports show that there are currently 39 million AIDS patients worldwide, 29.8 million of whom are on antiretroviral treatment, 1.3 million new HIV infections in 2022, and 630,000 deaths from AIDS-related illnesses (https://thepath.unaids.org/). The “One Bug-One Drug” approach for developing antiviral agents has achieved multiple remarkable successes in the ongoing battle against viruses [1]. Yet, in recent years, the rise of drug resistance and the unforeseen devastation wrought by emerging viruses, like SARS-CoV-2, have highlighted the potential limitations of the “One Bug-One Drug” approach in tackling the escalating variety of infections caused by unidentified pathogens [2, 3]. Therefore, exploiting novel and effective antivirals is still an urgent task.
Viruses may be extensively divided into two categories: enveloped viruses and non-enveloped viruses, based on whether they possess a lipid-rich envelope external to their protein capsid [4]. As the virus matures, it acquires its envelope from the cytoplasmic or nuclear membrane of the host cell, incorporating glycosylated proteins that are crucial for facilitating viral membrane fusion [5, 6]. Fusion inhibitors work in the early phase of membrane fusion during enveloped virus infection. Fusion inhibitors block the membrane fusion of the viral envelope with the host cell membrane or endosomal membrane by inhibiting the conformational changes of the fusion protein [7]. Unlike other antiviral drugs that inhibit viral replication inside host cells, fusion inhibitors are formulated to act outside the cell, focusing on the early stages of viral replication, like viral fusion. The advantage of this approach is that the therapeutic inhibitor does not need to penetrate the cell membrane, thereby minimizing potential harmful interactions between the viral pathogen and host cells [8]. The fusion inhibitors consist mainly of peptides, antibodies, small-molecule chemicals, and so on. Our research paper is dedicated to exploring fusion inhibitors that utilize peptides as their foundation [9, 10].
2. C-peptide fusion inhibitors
2.1 Origins and mechanism
The peptide-based fusion inhibitors originated in the HIV-1 field. Generally, HIV-1 attaches to host cell membrane and envelope protein subunit gp120 then binds to the cell receptor CD4+ and the co-receptor CXCR4 or CCR5, triggering transmembrane subunit gp41 conformational changes. Insertion of the gp41 N-terminus into the host cell membrane causes the C-terminal heptad repeat (CHR) region and the N-terminal heptad repeat (NHR) region to stretch into an intermediate state. Subsequently, the CHR region further interacts with the NHR region at the outside to form Six-helix bundle (6-HB) structure, which draws the viral envelope fusion with the host cell membrane, and facilitates fusion hole formation. Viral genetic material eventually enters the host cell through the fusion hole [11]. Compounds, which could inhibit antiviral activity by preventing endogenous 6-HB formation through binding with the CHR or NHR region, are generally referred to as fusion inhibitors [12, 13]. C-peptide fusion inhibitors, the most common peptide-based fusion inhibitor, sequences deriving from the gp41 CHR region or de novo designed, with the single-helix binding to gp41 NHR region, thus inhibiting 6-HB formation and exerting anti-HIV-1 ability (Figure 1) [14].
Figure 1.
The inhibitory mechanism of membrane fusion for C-peptides and N-peptides.
2.2 C-peptide modification strategies
Enfuvirtide (also known as T20) is the first listed C-peptide fusion inhibitor against HIV-1, which plays an important role in the AIDS treatment. However, in the clinical application process, T20 has shown major deficiencies in clinical application such as high level drug resistance, short half-life in vivo [15, 16]. And to date, researchers have traveled many miles to obtain novel peptide-based fusion inhibitors, but there is still no new drug that solves the above problems [17, 18]. Here, we have summarized six latest C-peptide modification strategies in order to provide reference for future candidate development.
2.2.1 The E-K salt bridge-based modification strategy
Peptide-based fusion inhibitors bind to the target generally with α-helical conformation. But the short peptide, as current hot candidate, tends to be randomly coiled, and thus the greater entropy loss for assembly into active α-helix during interaction with the target. Salt bridges formed between side chains positioned at intervals of
Figure 2.
The peptide sequences with E-K salt bridge (X represents norleucine amino acid residue).
The C34 peptide, exclusively derived from the HIV-1 gp41 CHR region [19], has demonstrated remarkable anti-HIV-1 activity, even maintaining its effectiveness against T20-resistant viral strains. However, compared to T20, C34 falls short as a candidate due to its lower solubility [20]. In 2002, Akira et al. obtained the peptide, named SC34 1, by replacing C34 residues located in the non-target binding region with hydrophilic glutamic acid (Glu) or lysine (Lys) to form the E-K salt bridge. SC34-1 not only overcame the solubility and α-helicity deficiencies of C34 but also exhibited a 10-fold increase in anti-HIV-1 activity compared to T20 [21]. Hiroki et al. further refined SC34-1 by reversing the E-K salt bridges at positions 16, 17, and 20, 21 in the sequence, resulting in SC34EK (Figure 2) [22]. The crystal structure of the SC34EK/target complex revealed that SC34EK adopted a standard α-helix due to the E-K salt bridges and had a propensity to form a heterologous 6-HB with the target peptide N36. The inhibitory activity of SC35EK against T20-resistant strains (including L33S, V38E, N43D, V38E/N42S) reached 0.9–4.3 nM, while T20’s inhibitory activity exceeded 1000 nM.
The E-K salt bridge modification has been proven to enhance α-helix formation and anti-HIV-1 activity, even conferring the ability to inhibit T20-resistant strains. Takeshi et al. shortened the SC34EK sequence to create SC29EK (Figure 2) [23]. SC29EK not only inhibited HIV-1 infection at a level comparable to SC34EK, but also blocked the replication of T20-resistant strains (EC50 = 2.7–4.1 nM) and maintained antiviral activity even in the presence of 50% serum concentrations. Hiroki et al. further truncated SC29EK to 22 peptide, named SC22EK (Figure 2) [24]. SC22EK currently stands as the shortest peptide employing the E-K salt bridge strategy. Experimental results indicated that SC22EK could suppress the C34-resistant strain (although data on T20-resistant strains was not available) and maintained significant anti-HIV-1 activity and high binding affinity. Therefore, designing peptides based on the E-K salt bridge strategy is a highly feasible approach that improves solubility, maintains the α-helix active conformation, and ultimately demonstrates potential inhibitory activity against the drug-resistant strains [25].
2.2.2 The stapled peptide-based modification strategy
Researchers have recognized that peptides derived directly from the natural sequences of fusion proteins often bind to their targets in secondary structures such as α-helices [26, 27, 28]. However, a significant challenge is that many peptide fragments are unable to form stable secondary structures and are prone to hydrolysis by peptidases, leading to reduced activity and bioavailability [29]. Fixation of the peptide helical structure by chemical modification has been shown to solve the above problems (Figure 3). In 2000, Verdine et al. introduced a method to stabilize the α-helical structure known as “Stapled Peptide.” This method utilizes carbon-carbon bonds as a scaffold to reinforce the helical conformation of the peptide, thereby enhancing its stability and potentially improving its bioactivity and resistance to enzymatic degradation [30]. By employing such strategies, researchers can develop more effective and durable peptide-based therapies for various diseases, including HIV-1.
Figure 3.
The schematic diagram of stapled modification strategy.
Inspired by the stapled modification strategy, Bird et al. obtained T20-1 and T20-2 by introducing the conformational stapled backbone at the
Figure 4.
Schematic diagram of stapled peptides (
2.2.3 The strategy for “M-T hook” introduction at the peptide N-terminus
The information provided describes an investigation by He et al. into the upstream motif of the C-terminal heptad repeat (CHR) peptide binding domain (PBD) region. They designed a peptide, CP621-652, that contains the “QIWNNMT” sequence at positions 621–627 of the CHR. This peptide interacts with a target peptide from the N-terminal heptad repeat (NHR) region to form a stable six-helix bundle (6-HB) structure. The “M-T hook” structure is a specific conformation formed by the conserved residues Met-626 and Thr-627 within the “QIWNNMT” sequence. Although most of the residues in this sequence are disordered in the electron density, Met-626 and Thr-627 fold into a quasi-hook shape that stabilizes the 6-HB structure (Figure 5). In this structure, Thr-627 alters the orientation of the peptide chain and positions Met-626 on top of a hydrophobic pocket. The side chain of Met-626 acts as a cover for the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. This stabilization contributes to the overall strength and stability of the 6-HB structure, making CP621-652 a more effective inhibitor of viral fusion than peptides lacking the “QIWNNMT” sequence [33, 34, 35].
Figure 5.
Crystal structure of the “M-T Hook” structure [34].
The research by He et al. and Chong et al. highlights the potential of the “M-T Hook” structure in optimizing HIV-1 fusion inhibitors. By mutating 11 residues in the CP621-652 peptide, He et al. obtained a novel 32-amino acid HIV-1 fusion inhibitor called CP32M. This peptide, which retains the “M-T Hook” structure, forms stable 6-HB complexes with the NHR region. CP32M can efficiently inhibit infection by several well-characterized T20-resistant viruses that carry an L33S point mutation or I37Q/V38Q, I37Q/V38M, and I37V/V38T double mutations; in sharp contrast, T20 had no inhibitory activity at a concentration as high as 750 nM [36]. Furthermore, Chong et al. obtained MT-C34 and MT-SFT by introducing “M-T Hook” at the N-terminus of C34 and Sifuvirtide, which showed stronger antiviral ability, and even the ability against T20-resistant strains [37]. Therefore, the “M-T Hook” structural modification strategy is an effective method to optimize the lead peptide, which keeps the peptide with highly antiviral activity, and may facilitate further sequence shortening. They also introduced the “M-T Hook” at the N-terminal of shorter peptides SC29EK and SC22EK, obtaining MT-SC29EK and MT-SC22EK, which retained high potency against T20- and SC29EK-resistant viruses (Figure 6) [38]. Based on the structural basis of MT-SC22EK, Chong et al. finally obtained a short peptide HP23 with 23 amino acid residues, which even exhibited picomolar anti-HIV-1 activity [39]. HP23 consists of “M-T Hook” structure, all PDB region and partial HBD region, tightly targeting the N-trimer of HIV-1 gp41 (Figure 6) [40]. HP23 was highly active in the inhibition of diverse HIV-1 subtypes, including T20 and MT-SC22EK-resistant HIV-1 mutants, and it exhibited a high genetic barrier to the development of resistance. The “M-T Hook” structure indeed provides more opportunities for peptides to interact with their targets and combat T20-resistant strains of HIV-1. This structural modification strategy has shown promising results in optimizing HIV-1 fusion inhibitors and enhancing their antiviral activity. Therefore, peptides incorporating the “M-T Hook” structure are expected to be further developed and tested as potential drug candidates for the treatment of HIV-1 infection.
Figure 6.
The peptide sequences containing “M-T Hook” portion.
2.2.4 The strategy for “IDL tail” introduction at the peptide C-terminus
It is well known that peptides containing “M-T Hook” structure in N-terminus showed anti-HIV-1, and even favored shorter sequences, so is it possible to introduce a particular structure at peptide C-terminus to obtain alternative novel short-peptide inhibitors? Guidance based on this question, researchers have identified another hook structure called the “IDL Tail” in the C-terminus of peptides. This structure is composed of isoleucine (Ile), aspartate (Asp), and leucine (Leu) residues and has been shown to interact with the NHR trimer of HIV-1 gp41 in a specific way. In the “IDL Tail” structure (Figure 7), the Ile and Leu hydrophobic side chains are oriented toward the NHR trimer and bind to the hydrophobic residues Leu545 and Val549. The Asp hydrophilic side chain targets the NHR through forming a salt bridge with the Asn553 side chain. Additionally, the Gln653 and Gln649 residues can form hydrogen bonds upstream of the “IDL Tail,” which further stabilize its structure.
Figure 7.
Crystal structure of the “IDL Tail” structure [41].
Inspired by the gp41-derived MT hook, Yun et al. synthesized the first short peptide against HIV-1, called CP-IDL, containing “IDL Tail” (Figure 8) [41]. The original CP peptide has low inhibitory activities against HIV-1 clinical strains, with IC50 of 281 and 207 nM, respectively, while CP-IDL showed the highest potency with increase of 109- and 55-fold, respectively. Also, CP-IDL could inhibit infection of all these T2635 or T20-resistant strains, which are much more potent than CP or T20. Furthermore, Chong et al. identified a shorter peptide [42], HP23, but found that it became ineffective against HIV-1 strains with the E49K mutation in the gp41 NHR region. However, by extending the C-terminus of HP23 with the “IDL Tail” structure, they created HP23-E6-IDL (Figure 8), which ameliorated the resistance to HP23 and showed increased inhibitory efficacy against HIV-1 strains and HP23-resistant mutants [43]. Compared to HP23, HP23-E6-IDL showed 2-fold to 16-fold increase in inhibitory efficacy against HIV-1 strains, and 12-fold increase in efficacy against HP23-resistant mutants. Subsequently, Su et al. synthesized other peptides, WQ-IDL and MT-WQ-IDL (Figure 8), by adding the “IDL Tail” structure and found that they showed more potent activities than T20 in inhibiting HIV-1IIIB infection [44]. In a later study, Su et al. introduced a mutation (T639I) into HP23-E6-IDL to generate the peptide YIK, which effectively inhibited HIV-1 infection and had a longer half-life [45]. While the peptides obtained using this strategy have not been fully investigated for their ability to inhibit T20-resistant strains, the results suggest that adding non-helical tails at the C-terminus may be a valid and universal strategy to improve the antiviral potency of C-peptide fusion inhibitors. As with the “M-T Hook” structure, further research and testing will be needed to evaluate the potential of “IDL Tail”-containing peptides as drug candidates for the treatment of HIV-1 infection.
Figure 8.
The peptide sequences containing “IDL Tail” portion.
2.2.5 The specific small-molecule conjugation strategy
We have known that, unlike peptides, small-molecule inhibitors boast cost-effectiveness and robust stability, rendering them highly favorable for pharmaceutical development [46, 47]. Within the realm of anti-HIV-1 fusion inhibitors, two small molecules—NB-2 and A12—have garnered attention for their micromolar-level activity against HIV-1 by targeting the PBD (Pocket Binding Domain) region of gp41 [20]. Notably, C34, a peptide harboring the PBD sequence, exhibits binding affinity toward the NHR (N-terminal Heptad Repeat) hydrophobic pocket, aligning with the specific target site of these small molecules [48]. Researchers have innovatively substituted the PBD segment of C34 with small molecules, thereby crafting small molecule-peptide conjugates (Figure 9). These conjugates harness a synergistic binding mechanism, where the small molecule and peptide portions lock onto distinct NHR regions. Wang et al. demonstrated this by excising the PBD portion of C34 to yield the peptide P26, and subsequently appending either A12 or NB-2 to the N-terminus of P26 (Figure 10) [49]. HIV-1 envelope protein-mediated cell-cell fusion assays have validated the superiority of these conjugates, exhibiting heightened inhibitory activity compared to their individual components or P26 alone. Remarkably, they effectively neutralized both T20-sensitive and T20-resistant HIV-1 strains, indicating that this specific conjugation strategy not only enhances antiviral efficacy but also exhibits drug resistance tolerance.
Figure 9.
The schematic diagram of specific small-molecule and peptide conjugate.
Figure 10.
The schematic of pyrrole-derived small molecule-peptide conjugate.
Saponins, a class of bioactive natural compounds primarily synthesized by plants as a defense mechanism against pathogens and herbivores, have demonstrated extensive antiviral properties. Notably, several pentacyclic triterpene saponins, including chikusetsusaponin IVa and glycyrrhizin, have exhibited broad antiviral activities [50]. Wang et al. synthesized alternative small molecule-peptide conjugates by linking saponin compounds to P26
Figure 11.
The schematic of saponins-derived small molecule-peptide conjugate.
In summary, the specific small-molecule peptide conjugation strategy offers a novel and effective approach to truncating long peptides into compact fragments while unlocking their therapeutic potential through coupling with specific small molecules. This innovative method holds promise as a means to address the limitations of both small molecules and long peptides by combining their respective advantages and mitigating their disadvantages. The resulting conjugates exhibit a robust synergistic effect, enhancing antiviral activity and metabolic stability, which could pave the way for the development of more potent and resilient therapeutic agents against HIV-1 and other viral infections.
2.2.6 The lipid small molecule conjugation strategy
The strategy of conjugating specific small molecules to the N-terminus of CHR-derived peptides, which bind to the NHR hydrophobic pocket domain, has been shown to enhance anti-HIV-1 activity and address issues of viral drug resistance. Additionally, researchers have identified other types of small molecules, such as cholesterol and fatty acids, that can substitute for the LBD portion in CHR-derived peptide sequences. These molecules facilitate the anchoring of peptide portion to the cell membrane, thereby increasing target region concentration (Figure 12) [51, 52, 53, 54, 55]. These small molecules could not specifically recognize NHR region, but rather enhance anti-HIV-1 capacity by enriching peptide concentrations in the membrane fusion region through non-specific effects such as lipophilicity, and are therefore considered “lipid-small molecule.”
Figure 12.
Putative mode of action of HIV-1 lipopeptides. The NHR domain is shown in the color column, the CHR domain is shown in the black spiral line, the non-lipopeptide is shown in the blue spiral line, and the lipopeptide is shown in the blue spiral line plus lipid molecule tail.
The integration of fatty acids into peptide scaffolds has emerged as a promising strategy for optimizing peptide-based fusion inhibitors, as demonstrated by Wexler’s early findings that fatty acid incorporation enhances antiviral activity by mimicking the LBD [56, 57, 58, 59, 60, 61]. Similarly, Hollmann et al. successfully linked cholesterol to the C34 sequence, resulting in a significant boost in anti-HIV-1 activity [58]. In 2016, Chong et al. synthesized a series of lipopeptides, among which LP-11, a fatty acid-conjugated lipopeptide comprising only 23 residues (Figure 13), exhibited potent and broad inhibitory activity against a diverse array of HIV-1 isolates and clinically resistant mutants. On this basis, Chong et al. further refined their approach by developing LP-19, a novel lipopeptide inhibitor with 23 residues (Figure 13), which incorporated multiple design strategies, including the M-T hook structure, partial HIV-2 sequences, and a membrane-anchoring lipid tail [62]. In vitro studies showed that T20, LP-11, and LP-19 inhibited T20-resistant mutants with mean IC50 values of 1804, 0.37, and 0.17 nM, respectively. Notably, LP-19 displayed significantly increased potency against both T20- and HP23-resistant viruses. In summary, LP-19 represents a promising candidate for clinical application development due to its potent antiviral activity, broad spectrum of activity, and resistance-breaking potential. This lipopeptide inhibitor holds significant promise for addressing the challenges posed by drug-resistant HIV-1 variants and improving the therapeutic options available for HIV-infected patients.
Figure 13.
The sequences of lipid-small molecule-peptide conjugate (Cholesterol, Palmitic acid).
In addition, we venture to speculate whether it is possible to use both specific and non-specific small molecules in combination with peptide couples. This approach, which encapsulates the targeting specificity of specific small molecules with the membrane-targeting ability of non-specific small molecules, has the potential to overcome the limitations of the above conjugates, providing a versatile and promising strategy for the development of potent antiviral agents.
3. N-peptide fusion inhibitors
3.1 Origins and mechanism
The membrane fusion process of class I viruses involves a complex interplay between the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) regions. During this process, the NHR regions undergo allosteric changes to form the N-trimer structural core. Subsequently, the CHR regions fold with the N-trimer to create a six-helix bundle (6-HB) structural domain. This 6-HB formation is crucial for viral fusion and entry into host cells. Current research has revealed that peptides sourced from the NHR domain are crucial for impeding the natural development of 6-HB by creating N-trimer mimetic structures that selectively attach to the CHR domain (Figure 1) [63]. Building on the previous point, N-peptides originating from the NHR sequences of class I viruses like HIV, MERS-CoV, and EBOV (Ebola Virus) have effectively modified the active conformation of the N-trimer and even demonstrated antiviral properties [12, 64, 65, 66, 67]. Consequently, the N-trimer functions as the essential foundation of viral fusion, and its significance in the fusion mechanism has paved the way for the identification of N-peptide lead compounds. These compounds directly reshape the native N-trimer or construct N-trimer mimics utilizing biochemical techniques. These discoveries have significant implications for the development of antiviral therapies targeting class I viruses, as they provide a potential means to disrupt the viral fusion process and prevent viral entry into host cells.
3.2 Two basic approaches to modify N-peptides
As previously stated, the two principal methods for modifying N-peptides are either mutating particular positions in the peptide sequences or integrating supplementary proteins or peptides into the peptide sequences. Each of these approaches aims to enhance the stability and inhibitory activity of the N-peptides. In the first approach, mutational modification involves altering specific amino acids in the peptide sequence based on the structure and function of the N-trimer molecule and the pattern of triple-helix formation. This can bolster the N-peptide’s spatial configuration stability and its capacity to interact with the target location, thereby augmenting its inhibitory effectiveness. As an example, Bewley et al. engineered N36Mut(e,g) through the substitution of nine amino acids at the specified (e.g.) site of N36. This mutant peptide exhibited increased inhibitory activity with a concentration of 308 nanomolar and functions by blocking the formation of endogenous 6-HB with the natural N-trimer [68]. The second approach involves embedding accessory proteins or peptides within the sequence to enhance the ability to assemble triple-helix structures. This can overcome the disadvantages of self-polymerization sediment and allow the novel N-peptides to form N-trimer mimics under suitable solution or physiological conditions. Debra et al. combined the instrumental peptide GCN4-pIQI’ with N36, N23, and N17 in the NHR region of HIV-1 gp41 to construct N-peptides’ chimeric variants (Figure 14). These chimeric peptides exhibited nanomolar levels of inhibitory activity, demonstrating the effectiveness of this approach in enhancing antiviral activity. Overall, both approaches have shown promise in improving the inhibitory activity of N-peptides against HIV-1.
Figure 14.
The architecture of HIV-1 gp41 and the design of chimeric N peptides. (A) An illustrative depiction of the primary structural arrangement of HIV-1 gp41. (B) Model of IQN17. (C) The sequence composition of the chimeric N-peptide [12].
3.3 The isopeptide bond bundling triple-helix for designing N-peptides
3.3.1 The creation of isopeptide linkages by incorporating tool peptides into the N-peptides
An isopeptide bond is a special kind of amide bond present in proteins or peptides, and it is defined by the inclusion of at least one amino or carboxyl side chain that is not located at the alpha position. This bond has been found to play a crucial role in stabilizing protein structures, as demonstrated by Kang et al. in their study of the Gram-positive bacterial pilus Spy0128 structure in 2007 [69]. Howarth’s team further investigated the mechanism of isopeptide bond formation and identified Lysine (Lys), Asparagine (Asn), Aspartic acid (Asp), and Glutamate (Glu) as key amino acids involved in this process [70, 71, 72]. The formation of isopeptide bonds can confer several beneficial characteristics to remodeled proteins or peptides, including chemical stability and protease stability [73]. Given these properties, the introduction of isopeptide bonds in the design of N-trimer mimics is a theoretically promising approach. By embedding isopeptide bonds within the supercoil framework, it might be feasible to create sturdy N-trimer imitations that display resistance to both chemical degradation and protease activity. This could lead to the development of more effective antiviral therapies that target the viral fusion process and prevent viral entry into host cells.
Initially, Lai’s team engineered two man-made peptides, referred to as 3HR and 4HR, with their sequences devised in accordance with the following principles (Figure 15): (i) By incorporating Isoleucine (Ile) residues at the (a, d) locations within the sequence, the peptides are enabled to autonomously assemble into a trimeric configuration [75]; (ii) The Glutamine (Gln) residues at the (b, c, f) positions within the sequence are utilized to enhance solubility; (iii) Arginine (Arg) and Glutamate (Glu) residues at the (g, e) position stabilize the trimer structure by electrostatic interaction in the inter-helical side chains; (iv) It is worth noting that alterations have been made to the
Figure 15.
A schematic depiction showcasing the generation of isopeptide bonds via an acyl transfer reaction occurring between helices, accompanied by a helical wheel representation of the N-terminal helical regions (NHRs) [74].
3.3.2 Creating isopeptide bonds via precise, location-specific mutations within the N-peptides
As has been noted, the primary approaches currently utilized for modifying N-peptides chiefly involve making targeted mutations at specific locations and integrating additional proteins or peptides. Lai’s research group has revealed that the peptide is capable of being linked by isopeptide bonds after the introduction of chimeric accessory peptides. Can isopeptide bonds be formed directly after introducing mutations at key sites in the natural sequence in order to stabilize the trimeric coiled-coil configuration? Certainly, the response is in the affirmative. By employing site-directed mutagenesis on the pivotal N36 sequence found in the HIV-1 NHR region, Lai was able to create N36 mutants such as N36M, N36MEK1, and N36MEK2. These mutants exhibit the capability to autonomously form trimeric coiled coils, and this is achieved without the requirement for chimeric auxiliary proteins or peptide chains [79]. Based on the sequence of N36, the 6th residue Gln at the
Figure 16.
The schematic N-peptide design for constructing isopeptide bonds based on site-specific mutations [81].
3.4 Breakthrough for designing broad-spectrum N-peptide inhibitors by isopeptide bond bundling triple-helix
Theoretically, for N-peptides to possess broad-spectrum antiviral activity, two essential prerequisites must be met: first, they must adopt a spatial conformation that is compatible with binding to the CHR (C-terminal heptad repeat) region of the viral envelope glycoprotein. Second, they must possess a sequence that, while potentially distinct from the natural NHR (N-terminal heptad repeat) region, still retains the ability to interact with the CHR domain in a spatially favorable manner. By utilizing the guidelines for constructing trimeric coiled coils and the mechanisms behind the creation of isopeptide bonds, N-peptides can be designed to be held together in a specific spatial arrangement by isopeptide bonds, resulting in the development of more stable imitations of N-trimers. These stabilized N-peptides, despite having sequences that differ from the natural NHR region, maintain interaction sites with the CHR domain and are capable of preventing viral membrane fusion. The distinct sequences of these covalently stabilized N-peptides may contribute to their broad-spectrum antiviral properties.
Wang et al. applied the coiled-coil design concept to modify N36 into N1G, followed by the incorporation of an isopeptide bond through an acyl transfer process, which led to the creation of the engineered peptide N3G. The results from the antiviral activity assessments indicated that N3G possesses an exceptionally broad antiviral spectrum, showing potent activity against HIV-1, MERS-CoV, the human pathogenic coronavirus OC43 (HCoV-OC43), and SARS-CoV-2 (Figure 17) [82]. These research findings broke through two significant challenges by constructing N-trimer supercoiling structure: (i) N-peptides exert high inhibitory activity against Human β-Coronavirus for the first time; (ii) N-peptides present broad-spectrum antiviral activity, breaking through virus species. Coronaviruses can infect host cells through plasma membrane pathway and endosomal pathway [83], while peptides are generally unable to enter the endosomes to exert inhibitory activity. Saskia et al. have disclosed that synthetic coiled coils, incorporating positively charged residues, are capable of traversing cellular membranes [84]. The mode of action for this coiled coil is presumably similar to that of cell-penetrating peptides (CPPs), in which the arginine (Arg) residues of CPPs strongly bind to acidic protein domains, sulfated glycans, and the hydrophilic head groups of membrane phospholipids present on the cell surface. This binding subsequently initiates the process of endocytosis [85]. Due to its coiled-coil configuration that features positively charged arginine (Arg) and lysine (Lys) residues, N3G is apt to interact with negatively charged molecular entities on the cell membrane’s exterior. Such an interaction may prompt endocytosis and hinder the fusion of viral membranes within the endosome, consequently showing promise as an inhibitor against coronaviruses. In terms of its wide-ranging antiviral capabilities across different virus types, N3G undergoes a process of three-dimensional folding and self-assembly, ultimately forming an N-trimer that contains binding sites characterized by strong affinity. N3G decreases the degree of conformational entropy loss when binding to the CHR region, allowing it to create heterologous 6-HB (six-helix bundle) complexes with the CHR regions of multiple viruses via non-specific hydrophobic interactions. As a result, N3G demonstrates broad-spectrum antiviral activity.
Figure 17.
The coiled-coil peptide against HIV-1 and Human β-Coronavirus infection [82].
4. Conclusion
The high pathogenicity and mutation susceptibility of viruses have driven researchers to continuously seek novel fusion inhibitors. The approval of peptide-based fusion inhibitors such as Enfuvirtide by the U.S. Food and Drug Administration (FDA) and Albuvirtide by the National Medical Products Administration (NMPA, previously known as CFDA) has paved the way for antiviral drug research [86]. Researchers remain committed to discovering more peptide-based lead candidates and understanding their molecular mechanisms. For instance, Sifuvirtide has already entered clinical trials in China [87]. The development of these inhibitors holds promise for addressing the ongoing threat posed by viruses, including those that have the potential to cause pandemics. By continuing to explore and refine peptide-based fusion inhibitors, researchers aim to create more effective and broadly applicable antiviral drugs that can protect public health and respond to emerging viral threats.
Acknowledgments
We thank the financial support from the Inner Mongolia Natural Science Foundation of China (No. 2023LHMS08045;2025QN08006), the ZHIXUE Talent Project of Inner Mongolia Medical University 2024–2026 (No. ZY20241203), the Laboratory Open Fund Project of Inner Mongolia Medical University (No. 2024GZ17;2024ZN26), the Sub-project of Quality Enhancement and Cultivation Discipline for the Pharmacy College of Inner Mongolia Medical University (No. YXY2024TZ701), and Basic Research and Applied Basic Research Projects in Hohhot (No. 2024-规-基-20).
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