1. Human Immunodeficiency Virus (HIV): Structure and Prevalence of Infection

2. Anti-HIV Agents

3. Peptide-Based Anti-HIV Agents

3.1. Peptide-Based Entry Inhibitors

The envelope surface of HIV-1 is a glycoprotein with a molecular weight of 160 kD (gp160), crucial for binding and facilitating viral entry into host cells. The gp160 gene, after translation, is cleaved into two noncovalently associated receptor-binding subunits, gp120, and a fusion protein subunit, gp41. Transmembrane gp41 anchors Env to the membrane and associates non-covalently with gp120, thereby forming a stable trimer of heterodimers, the metastable Env prefusion conformation. When gp120 binds to the cellular receptor CD4 and a chemokine coreceptor, it initiates intricate conformational changes in gp41, ultimately resulting in the fusion of viral and cellular membranes.

The ectodomain of gp41 consists of several functional regions: the N-terminal fusion peptide, heptad repeat 1 (HR1, N-heptad repeat, N-terminal helices (NHR)), and heptad repeat 2 (HR2, C-terminal helices (CHR)), along with the transmembrane region (TMR). The fusion-active (fusogenic) conformation of gp41 is a six-helix bundle, where three NHR assemble into a central trimeric coiled coil, while three CHR interlock in an antiparallel fashion within the hydrophobic grooves of this coiled core [19] (Figure 2). N peptides assemble into a central trimeric coiled coil (N trimer), forming grooves where C peptides can bind. The N- and C-terminal heptad regions of the gp41 are attractive targets for designing anti-HIV agents [19,20]. Concerning peptide inhibitors, the majority of synthetic peptides investigated for targeting the entry stage of the HIV-1 life cycle originate from various domains of the HIV-1 glycoprotein gp41.

The gp120 subunit features five conserved constant regions (C1-C5) and five variable regions (V1-V5), which play essential roles in initiating and regulating HIV-1 infection. The interaction between the host receptor CD4 and specific residues in the conserved regions of gp120 occurs on either side of the V4 region (Figure 3).

Although CD4 was quickly recognized as the ‘primary receptor’ for the AIDS virus, it soon became evident that other molecules might also play a role. CCR5 and CXCR4 are also essential for facilitating the necessary conformational changes during the early stages of the viral cycle. Both CCR5 and CXCR4 are chemokine receptors structurally related to one another and belong to the superfamily of seven-transmembrane G-protein coupled receptors (GPCRs) [21].

The coreceptor CCR5 interacts with gp120, particularly with a GPGR/Q motif located at both the apex and base of the V3 loop. The spike-exposed surface of gp120 is primarily characterized by its variable regions and a high density of carbohydrates. These highly variable regions and the carbohydrate coating contribute to the protein's extracellular interactions [22].

Various antibodies have been developed to target these conserved regions, including the V3 glycan attached to N332 glycoprotein, the CD4-binding site (CD4bs), and the V1-2 loops. Key interactions include the binding of the gp120 V3 loop to the chemokine binding pocket on CCR5 and the engagement of the CD4-induced bridging sheet of gp120 with the N-terminus of CCR5. This structural information is crucial for designing optimized inhibitors [23].

Figure 3. The steps of gp120 emergence for the initiation and regulation of HIV-1 infection. Glycoprotein gp160 is cleaved into gp120 and gp41 despite being attached in a trimeric form on the viral surface. The gp41 C-terminal subunit undergoes a conformational change necessary for viral fusion, while the gp120 N-terminal subunit extends outside the viral membrane. Gp120 can be structurally organized into five conserved regions (C1-C5) and five variable regions (V1-V5). The interaction between the host receptor CD4 and specific residues in the conserved regions of gp120, along with the coreceptor CCR5 binding to a GPGR/Q motif, is critical. The variable regions of gp120 and a significant amount of carbohydrates contribute to extracellular interactions and cover the protein's surface [22]. Figure 3 was created with BioRender.com.

Figure 3. The steps of gp120 emergence for the initiation and regulation of HIV-1 infection. Glycoprotein gp160 is cleaved into gp120 and gp41 despite being attached in a trimeric form on the viral surface. The gp41 C-terminal subunit undergoes a conformational change necessary for viral fusion, while the gp120 N-terminal subunit extends outside the viral membrane. Gp120 can be structurally organized into five conserved regions (C1-C5) and five variable regions (V1-V5). The interaction between the host receptor CD4 and specific residues in the conserved regions of gp120, along with the coreceptor CCR5 binding to a GPGR/Q motif, is critical. The variable regions of gp120 and a significant amount of carbohydrates contribute to extracellular interactions and cover the protein's surface [22]. Figure 3 was created with BioRender.com.

Figure 4 shows the mechanism of action and the advantages of peptide-based HIV entry inhibitors, which interact with various sites on either viral surface proteins or cellular receptors. These include peptides that target the CD4-binding site on gp120, co-receptors, the co-receptor-binding site on gp120, the fusion peptide (FP) in gp41, and peptides that target the CHR and NHR regions in gp41. These inhibitors act in the early stage of viral infection. Additionally, other peptide-based anti-HIV strategies have been developed to neutralize cell-free virions [24].

3.1.1. Peptide-Based HIV Entry Inhibitors Targeting gp120 or gp41

The roles of gp120 and gp41 in the HIV lifecycle have greatly influenced the development of anti-HIV peptides. The U.S. Food and Drug Administration (FDA) approved enfuvirtide (ENF, T-20, Fuzeon) and albuvirtide as peptide-based anti-HIV drugs in 2003 and 2018, respectively (Figure 5). ENF, the first peptide drug to inhibit HIV entry, was approved for patients unresponsive to other antiretrovirals [23]. It works by binding to the gp41 protein on the HIV envelope, blocking the required conformational changes for membrane fusion and viral entry [25,26]. ENF binds to helical regions in the viral gp41 protein, obstructing the formation of the six-helix bundle essential for the fusion of the viral and host cell membrane. ENF is a C peptide that binds to a portion of the N-trimer groove, preventing the formation of the six-helix bundle in a dominant-negative manner. More specifically, ENF attaches to the first heptad repeat (HR1, N-helix) within the gp41 subunit of the viral envelope glycoprotein. Effective against various HIV-1 strains but not HIV-2, ENF requires high dose twice-daily intravenous administration due to its short half-life [7]. It can cause uncomfortable injection-site reactions and is quite expensive [9].

The pharmacokinetics of ENF were improved through chemical glycosylation. This approach leverages the protective effects of glycosylation against protein denaturation and protease degradation. Glycosylated ENF, specifically with sialic acid (SL-ENF), demonstrated activity against HIV [27]. Additionally, to address ENF's poor aqueous solubility, it was conjugated with polyethylene glycol (PEG). This conjugation resulted in increased solubility and an extended half-life while maintaining similar antiviral activity against HIV-1 (EC₅₀ = 6–91 nM). The PEGylated form showed a high affinity for a functional domain of the HIV gp41 protein, effectively inhibiting the gp41-mediated fusion between the virus and host-cell membranes [28].

Conversely, albuvirtide (ABT, FB006M) is another HIV-1 fusion inhibitor that functions as a long-acting fusion inhibitor by attaching to the transmembrane glycoprotein gp41 on the outer membrane of HIV. ABT is a peptide modified with 3-maleimidopropionic acid, and it is derived from the N-terminal sequence of HIV-1 gp41. It prevents replication by disrupting the fusion between the virus and the host cell membrane. ABT binds to the HR1 domain of gp1 [29] and has a significantly longer half-life compared to T20, which requires administration through injections twice daily. ABT demonstrates broad-spectrum anti-HIV-1 activity in vitro, with a half-maximal inhibitory concentration (IC₅₀) ranging from 0.5 to 5.0 nM. It has proven effective against 28 distinct clinical isolates of HIV-1 in China, exhibiting IC₅₀ values between 1.3 and 18.1 nM [30].

Several other peptide sequences have been investigated (Table 2) to interfere with HIV entry and effectively inhibit HIV infection. Pu et al. (2019) have reviewed some of these peptides. We have organized the classes and extracted several sequences in addition to the previously reported peptides.

Various peptide-based inhibitors target sites like gp120 CD4-binding site (CD4bs), coreceptor binding site (CoRbs), and gp41 regions such as fusion peptide (FP), N-heptad repeat (NHR), and heptad repeat (CHR) (Figure 2). For instance, CD4M33 and M48U1 (Table 2) target the CD4-CD4bs on the HIV-1 envelope with IC₅₀ values of 424.7 and 0.71 nM, respectively. The main peptide-based HIV entry inhibitors targeting gp120 or gp41 are discussed below:

3.1.1.1. Inhibitors Targeting gp41 N-heptad repeat (NHR) Interactions

The conserved N-heptad repeat (NHR) region of gp41, which is formed after binding between the CD4 receptor and HIV-1 envelop spike (Env), is one of the main targets of designing peptide inhibitors. Some of the reported gp41 NHR inhibitors peptides are SJ-2176, T20, C34, SFT, SC29EK, T1249, T1144, FB006M (ABT), CP32M, HP23, AP3, HP23-E6-IDL, YIK-C16, and MT-WQ-IDL (Table 2) with IC₅₀ values of 101.0, 29.5, 12.5, 50, 9.6, 3.4, 13.9, 2.7, 65.0, 4.7, 19, 0.75, 0.09, and 2.7 nM, respectively. Also, LP11, LP40, LP46, LP52, and C34Chol (Table 2) were screened as gp41 NHR inhibitors peptides and showed IC₅₀ values of 0.83, 4.29, 0.08, 0.017, and 15.5 nM, respectively.

The synthetic peptide C34 has the amino acid sequence WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL (see Table 2) and features N-terminal acetylation and C-terminal amidation for enhanced stability. Two PEGylated variants of C34, PEG2kC34, and PEG5kC34, were tested for their anti-HIV properties. Their effectiveness in inhibiting HIV-1 Env-mediated cell-cell fusion was evaluated using CD4 and coreceptor-expressing TZM-bl cells as targets and HIV-1 Env-expressing HEK293T cells as effectors. The findings revealed that PEGylated C34 peptides demonstrated broad-spectrum anti-HIV-1 activity with a prolonged half-life, achieving EC₅₀ values of approximately 36 nM for both PEG2kC34 and PEG5kC34. This indicates that PEGylation could be a promising strategy for developing long-acting peptide-based anti-HIV drugs [31].

He et al. (2023) developed lipopeptide-19 (LP-19) with a sequence of EMTWEEWEKKVEELEKKIEELLK-PEG8-K(C16) as a broad-spectrum anti-HIV fusion inhibitor effective against HIV-1 and HIV-2, with a high resistance barrier. This peptide was designed by adding a fatty acid group, palmitic acid (C16), to the C-terminus of the peptide 2P23, aiming to target a highly conserved pocket site on the HIV-1 gp41 protein using the M-T hook structure (Figure 2). The IC₅₀ values of LP-19 against various HIV subtypes were 0.76 nM for subtype B, 0.29 nM for CRF01_AE, 0.38 nM for CRF07_BC, 0.85 nM for CRF08_BC, and 0.44 nM for URF [32].

As described earlier, ENF (Figure 5) is a peptide drug used in combination therapy to treat HIV-1 infection, acting as a membrane fusion inhibitor. While ENF has proven effective in combination therapies, its limited antiviral activity and the potential for resistance have driven efforts to enhance its efficacy. Recent research has focused on conjugating T-20 backbone-based fusion inhibitors with lipids to boost its anti-HIV activity. For example, cholesterol-conjugated peptides LP-83 and LP-86, depicted in Figure 6, demonstrated strong inhibitory effects against various HIV-1 strains, including HIV-1NL4-3 (X4 tropic), HIV-1JRCSF (R5 tropic), and HIV-189.6 (R5X4 tropic), with IC₅₀ values of 0.49 ± 0.03, 4.54 ± 1.42, and 4.75 ± 0.83 pM for LP-83, and 0.43 ± 0.03, 4.78 ± 0.69, and 7.24 ± 1.36 pM for LP-86, respectively[33] .

Short peptide-based inhibitors of HIV-1 fusion have been developed by creating a series of m-xylene thioether-stapled 22-residue α-helical peptides using a dithiol bisalkylation reaction. These peptides target the HIV-1 glycoprotein gp41 and act as fusion inhibitors. By focusing on the helix-zone binding domain of the gp41, researchers successfully synthesized stapled peptides, including one named hCS6ERE. The peptide Hcs6ERE has the sequence IEELI^A AQ^QQRK NEEALRE L, where ^ indicates the positions of homocysteine residues that form staples upon reaction. This peptide demonstrated potent inhibition of HIV-1, with an EC₅₀ of 7.2 ± 0.3 nM, effectively blocking env-mediated cell-cell fusion and viral replication. Furthermore, combining Hcs6ERE with another fusion inhibitor, HP23, which targets NHR region, resulted in a synergistic anti-HIV-1 effect. Both peptides target the gp41 NHR, with HP23 overlapping a 6-amino acid sequence at the N-terminus of Hcs6ERE. HP23 features an M-T hook structure and a PBD sequence, primarily binding to the pocket region on the gp41 NHR [34].

In a separate study by Mzoughi et al. (2019), two trimeric synthetic peptides—N36 (546SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL581) and C34 (628WMEWDREINNYSTLIHSLIEFSQNQQEKNEQELL661)—mimicked the N and C-terminal heptad repeats HR1 and HR2 domains, which are essential for HIV-1 entry through membrane fusion, were evaluated. The research showed that trimerization of the N36 peptide enhanced both its stability and antiviral activity, whereas trimerization of the C34 peptide did not significantly improve its stability or efficacy. The improved performance of the trimeric N36 peptide was attributed to its C-terminal region better mimicking the flexible orientation of this region in the HIV-1 gp41 structure. Conversely, the addition of two amino acids, arginine and glutamic acid, at the C-terminal end of the C34 peptide (C34 RE) enhanced its activity. Additionally, the study found that coupling trimeric peptides with various carriers capable of inducing coiled-coil trimerization and maintaining α-helix structures preserved, or even enhanced, their antiviral activity compared to monomers. These results underscore the potential of trimeric peptides as HIV-1 inhibitors and suggest promising directions for developing peptide-based fusion inhibitors [35].

In summary, peptide inhibitors targeting the NHR region of HIV-1 gp41 have demonstrated significant potential in blocking viral entry and fusion. Recent advancements have significantly enhanced the efficacy and stability of these inhibitors. For instance, PEGylated versions of the C34 peptide, such as PEG2kC34 and PEG5kC34, showed broad-spectrum anti-HIV-1 activity and prolonged half-lives, highlighting PEGylation as a promising strategy for developing long-acting anti-HIV drugs. Additionally, lipopeptide-19 (LP-19) and cholesterol-conjugated peptides LP-83 and LP-86 exhibit inhibitory effects against multiple HIV-1 strains, suggesting that lipid conjugation can further enhance peptide efficacy. Overall, these developments underscore the continuing relevance of peptide inhibitors targeting the NHR region of gp41. Innovative approaches, such as the use of stapled peptides and trimeric constructs, have also shown promising results. Stapled peptides like Hcs6ERE exhibit strong antiviral activity, while trimeric peptides, such as N36, demonstrate improved stability and efficacy due to their structural mimicry of the HIV-1 gp41 heptad repeat regions. The combination of enhanced peptide stability, novel conjugation techniques, and strategic structural modifications paves the way for more effective and durable anti-HIV-1 therapies.

3.1.1.2. Inhibitors Targeting gp41 Heptad Repeat (CHR) Interactions

A number of peptides were also based on the carboxy-terminal leucine/isoleucine heptad repeat (CHR) region of gp41 (Table 2). Peptides T21/DP107 [36,37] and IZN17 [38] (Table 2) inhibited the gp41 CHR region with IC₅₀ values of 320 and 22 nM, respectively.

Several other peptide inhibitors targeting HIV-1 entry were designed to match the C-terminal heptad repeat of the HIV-1 gp41 protein (C-peptides). These peptides function by binding to the central coiled-coil region of gp41 in a helical conformation, acting in a dominant-negative manner. Among these, C14linkmid and C14Aib [39] (Table 2), which showed minimal helical content or propensity, exhibited inhibitory potency with IC₅₀ values of 35 µM and 144 µM, respectively, and strong binding affinity to the gp41 hydrophobic pocket. The effectiveness of these peptides is significantly influenced by the position of the crosslinker within the peptide sequence. An N-terminal crosslinker, for instance, incurs a greater enthalpic penalty, leading to reduced binding affinity. The incorrect positioning of crosslinkers, such as the misalignment of Trp-628 and Trp-631, which are key pocket-binding residues, also impacts efficacy. Specifically, Trp-628, being outside the linker of C14linkmid, contributes to entropic stabilization by hindering the crosslinker's ability to cap the helix. Consequently, stabilization at the N-terminal, which is generally more flexible than the central part of the helix, results in a more significant reduction in degrees of freedom compared to stabilization in the middle of the helix [39].

Wild et al. (1994) synthesized peptides based on CHR HIV-1 gp41 protein. Peptide DP-178 was derived from residues 643-678 of the HIV-1_LAI isolate and designed to adopt an α-helical secondary structure. The peptide inhibited the HIV life cycle at an early stage before reverse transcription. DP-178 (YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF) demonstrated inhibitory effects against primary HIV-1 strains 596, 598, and LA1 in PBMCs, with ID₅₀ (dose of peptide causing a 50% reduction in reverse transcriptase cpm) values of 2.8 µg/ml, 1.2 µg/ml, and 1.1 µg/ml, respectively. These results suggested that DP-178 exerts its antiviral effect by interfering with viral entry or uncoating, affecting early steps before provirus formation, rather than later stages involving envelope synthesis, processing, or assembly [40].

The combination of natural α-amino acids with unnatural β-amino acids results in a distinctive class of molecules known as α/β-peptides. These α/β-peptides possess a mixed backbone that supports a variety of folding patterns and biological functions, similar to natural peptides and proteins. Despite their unconventional structures, α/β-peptides offer notable advantages over natural peptides, such as enhanced stability against protease-mediated degradation [41]. By integrating both α- and β-amino acids into polypeptide sequences derived from the HIV protein gp41, Horne et al. (2009) created molecules that effectively replicate the structural and functional properties of critical gp41 subunits. These α/β-peptides demonstrated to inhibit HIV-cell fusion through mechanisms akin to those of gp41-derived α-peptides, highlighting their potential as inhibitors in HIV research. The α-peptide structure illustrated in Figure 7 binds tightly to gp41-5, demonstrating significant inhibition of HIV-1 infectivity, with IC₅₀ values ranging from 5 ± 0.6 to 56 ± 6 nM depending on the strain. The cell-cell fusion assay results demonstrated that the two α/β-peptides, Ac-TTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL-NH₂ and Ac-TTWEXWDZAIAEYAXRIEXLIZAAQEQQEKNEXALZEL-NH₂, exhibited IC₅₀ values of 7 ± 2 nM and 5 ± 2 nM, respectively. These findings indicate that these peptides effectively mimic the structural and functional aspects of gp41 through their CHR-derived α/β-peptide sequences [42].

In summary, peptides targeting the CHR region of the HIV-1 gp41 protein have shown varying degrees of effectiveness in inhibiting viral entry. While peptides targeting the CHR region of gp41 offer promising therapeutic potential, their effectiveness is closely linked to their structural design and binding characteristics. The introduction of α/β-peptides, which combine natural α-amino acids with unnatural β-amino acids, presents a promising alternative due to their enhanced stability and effective mimicry of gp41 structures. Continued optimization of these peptides, including precise crosslinker placement and structural modifications, is essential for enhancing their inhibitory potency and advancing their application in HIV treatment.

Table 2. Peptide-based HIV entry inhibitors targeting gp120 or gp41 interactions.

Table 2. Peptide-based HIV entry inhibitors targeting gp120 or gp41 interactions.

Inhibitors Targeting gp120 CD4 Binding Site Interactions
CD4M33	TpaNLHFCQLRCKSLGLLGKCAGSBipCACV-NH2 Tpa = Thiopropionic acid; Bip = biphenylalanine.	[43]
M48U1 M48U2 M48U3		[44]; [45]
Inhibitors Targeting gp41 N-heptad repeat (NHR) Interactions
SJ-2176	Amino-acid residues 637-666: EWDREINNYTSLIHSLIEESQNQQEKNEQEGGC	[46]; [47]
T20 (ENF)	YTSLIHSLIEESQNQQEKNEQELLEDKWASLWNWF	[48]
C34	WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL	[49]; [31]
PEG2kC34 and PEG5kC34	Pegylated variants of C34	[31]
C34-cholesterol (C34-Chol)	C34-GlySerGly-Cys(Chol)	[50]
CP34 RE	CP34-Arg-Glu	[35]
SFT		[51]
SC29EK		[52]
T1249	WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASL WEWF	[53]
T1144	TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALREL	[54]
FB006M (ABT)		[55]; [29]
CP32M	i to i + 4 position of the helical conformation.	[56]
HP23	EMTWEEWEKKIEEYTKKIEEILK	[57]
AP3	KKISEEQKKIQEEIKKILEESKKILEEIKKDWEEWIM	[58]
HP23-E6-IDL (626-656)	EMTWEEWEKKIEEYTKKIEEILKKSQNQQIDL IDL = Ile-Asp-Leu)	[59]
YIK-C16	EMTWEEWEKIEEYIKKIEEILKKSQNQQIDLGSG-PEG4-K(Palm)	[60]
MT-WQ-IDL (626-656)	MTWEEWDKKIEEYTKKIEELIKKSQNQQIDL	[61]
LP-11	EMTWEEWEKKIEEYTKKIEEILK-PEG8-K(C16)	[62]
LP-19	EMTWEEWEKKVEELEKKIEELLK-PEG8-K(C16)	[32]
LP40	YTSLIHSLIEESQNQQEKNEQELLELDK(C16)	[62]
LP46	WQEWEQKI-------TALLEQAQIQQEKNEYELQKLDK(C16)	[63]
LP52		[63]
LP-83	WEQKIEELLKKAEEQQKKNEEELKKLEKC(Chol)	[33]
LP-86	LEANIEELLKKAEEQQKKNEEELKKLEKC(Chol)	[33]
Hcs6ERE	IEELI^A AQ^QQRK NEEALRE L	[34]
N36	SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL	[35]
Inhibitors targeting gp41 heptad repeat (CHR) Interactions
T21/DP107	Ac-NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQ-NH2	[36]; [37]
IZN17	Ac-IKKEIEAIKKEQEAIKKKIEAIEK...............LLQLTVWGIKQLQARL-NH2	[38]
C14linkmid		[39]
C14Aib		[39]
DP-178	YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF	[64]
CHR-derived α/β-peptide sequences	AcTTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALREL-NH2 AcTTWEXWDZAIAEYAXRIEXLIZAAQEQQEKNEXALZEL-NH2	[42]
Inhibitors Containing a Pocket-Binding Domain
P35A4	QEESIKKWEEWSKKIEELIKKSEELIKKIEEQIKK	[65]
PP24C	QEESIKKWEEWSKKIEELIKKIEEQIKK-PEG24(NH2-(PEG)24-CH2CH2COOH)-C(Chol)	[65]
PIE-12		[66]
Inhibitors Targeting Coreceptor CXCR4 or CCR5 Binding Interactions
pV2α-Tys	Lys-Val-Gln-Lys-Glu-Tyr(SO3H)-Ala-Leu-Phe-Tyr(SO3H)-Glu-Leu-Asp-Ile-Val-Pro-Ile-Asp	[67]
pCCR5-Tys	Met-Asp-Tyr-Gln-Val-Ser-Ser-Pro-Ile-Tyr(SO3H)-Asp-Ile-Asn-Tyr(SO3H)-Tyr-Thr-Ser-Glu-Pro-Ser-Gln-Lys	[67]
Cyclic disulfide peptides (L and D)		[68]
EPI-X4 JM#173-C	d-I-LRWSRKC	[69]
Trifunctional construct	SAv-VIR-102C9-EPI-X4	[69]
Irreversible Env Inactivators
Macrocyclic peptide triazole AAR029b		[70]
FITC-AAR029b		[70]
UM15		[70]
Inhibitors Targeting gp41 Fusion Peptide (FP) Interactions
VIR-576 VIR-353	(LEAIPCSIPPEFLFGKPFVF)×2 LEAIPCSIPPCFLFNKPFVF	[71]; [72]
E1P47 E1P47-1 E1P47-2	WILEYLWKVPFDFWRGVI ILEYLWKVPFDFWRGVIS LEYLWKVPFDFWRGVISL	[73]
Transmembrane protein sequence-derived anti-HIV peptides
P3	WQEWEQQVRYFLEANISQRLEQAQIQQEKNMYELQKLNSWDVFGNWF	[74]

3.1.1.3. Inhibitors Containing a Pocket-Binding Domain

The development of HIV-1 entry inhibitors has also focused on peptides containing a pocket-binding domain (PBD) designed to target the deep pocket region and adjacent subpocket sites on the N-trimer of the HIV-1 gp41 protein, which are critical for HIV-1 fusion. Notable examples include the artificial α-helical peptides P35A4 (QEESIKKWEEWSKKIEELIKKSEELIKKIEEQIKK) and PP24C (QEESIKKWEEWSKKIEELIKKIEEQIKK-PEG₂₄(NH₂-(PEG)₂₄-CH₂CH₂COOH)-C(Chol)), with PP24C being cholesterol-tagged [65]. The α-helical complexes formed between PP24C and the NHR peptide T21 or N36 are comparable to those formed with the parent peptide P35A4. Specifically, the interaction between PP24C and T21 forms a heterogeneous 6HB complex. These peptides inhibit HIV-1 infection by binding to the exposed viral gp41 NHR region and disrupting the formation of the 6-HB complex between gp41 NHR and CHR. The binding of T21 to P35A4 or PP24C targets both the gp41 deep cavity and subpocket sites, effectively inhibiting HIV-1 fusion [65].

The HIV gp41 N-trimer pocket region is an optimal target for viral intervention due to its extracellular location, high conservation, and crucial role in viral entry. The D-peptide PIE-12 trimer binds explicitly to this pocket region of gp41. Additionally, D-peptides are more resistant to protease degradation. Welch et al. (2010) discovered a pocket-specific D-peptide using phase display as PIE12 (HPCDYPEWQWLCRLGK) which contains a short core sequence surrounded by a disulfide bond between two cysteine residues that imparts structural rigidity required for binding. The analog showed IC₅₀ values of 33 nM and 1100 nM against HXB2 and JRFL strains, respectively. PIE12-monomer was generally much less potent than PIE12-trimer. The PIE12-trimer could endure resistance mutations that affected earlier D-peptides, requiring a significantly longer selection period (65 weeks) to develop resistant strains [66].

3.1.1.4. Inhibitors Targeting Coreceptor CXCR4 or CCR5 Binding Interactions

HIV-1 variants rely on either CCR5 or CXCR4 for infection, with CCR5 being crucial for initial transmission and chronic infection. Maraviroc (Selzentry) is a non-peptidic approved medication that inhibits the CCR5 co-receptor on the host cell surface, but it is ineffective against HIV-1 strains that utilize the CXCR4 receptor for viral entry [75].

Inhibitors pV2α-Tys and pCCR5-Tys were identified by Cimbro et al. (2016) as interacting with gp120 coreceptor binding sites (CoRbs), exhibiting IC₅₀ values ˂50,000 and ˃50,000 nM, respectively. The tyrosine-sulfated peptide pV2α-Tys, derived from the V2 region, mimics the CCR5 N-terminus both structurally and functionally, effectively blocking HIV-1 infection. This peptide adopts a CCR5-like helical conformation, allowing it to interact directly with gp120 in a CD4-dependent manner, and competes with the CCR5 N-terminal peptide [67].

While sulfated V2 mimics show promise, neither sulfated nor non-sulfated V2 peptides significantly inhibited HIV-1 entry and fusion by blocking coreceptor utilization. The key factor appears to be the highly conserved C-terminal sulfotyrosine (Tys177), which plays a dominant role in their effectiveness. The ability of CCR5 N-terminal peptides and V2 mimics to inhibit a broad range of HIV-1 strains, regardless of coreceptor tropism, underscores the importance of the overall structural conservation of the gp120 coreceptor-binding site [67].

During HIV entry, the third variable loop (V3 loop) of the HIV-1 envelope glycoprotein gp120 binds to the CXC chemokine receptor 4 (CXCR4) coreceptor. To investigate this interaction, synthetic peptides covering the entire V3 loop were used by Zhu et al. [68]. By creating a cyclic peptide with enhanced conformational stability, the ends of the V3 loop were covalently linked via a disulfide bond (Figure 8). To further study the impact of peptide side-chain conformations on CXCR4 recognition, an all-D-amino acid analog of the L-V3 loop peptide was synthesized. Both cyclic L- and D-V3 loop peptides exhibited similar binding to the CXCR4 receptor, but not to the CCR5 chemokine receptor, demonstrating selective interaction with CXCR4. These results suggest that the HIV-1 gp120 V3 loop-CXCR4 interface can accommodate ligands with different chiralities, potentially allowing the virus to maintain coreceptor recognition even with V3 loop mutations [68].

The gp41 fusion peptide has been targeted by a virus-inhibitory peptide (VIRIP), a 20-amino acid fragment derived from α1-antitrypsin. The endogenous peptide inhibitor of CXCR4 (EPI-X4), a 16-residue fragment of human serum albumin, blocks HIV-1 entry by binding to the CXCR4 co-receptor. Two promising peptides, VIRIP102C9 (sequence LEIAIPMSICEVPPNKPFVF), a fusion peptide inhibitor, and EPI-X4 JM#173 (sequence d-I-LRWSRKC), an optimized variant of EPI-X4 as CXCR4 antagonist, were evaluated for their potential. Combining these peptides into a single construct (streptavidin (Sav)[13]-VIR-102C9-EPI-X4 JM#173-C) demonstrated enhanced activity against both CCR5- and CXCR4-tropic HIV-1 variants. The study highlighted the benefits of integrating three different thiol-reactive moieties into a single system using a bis-sulfone moiety coupled with a maleimide functionality. Additionally, combining the two peptide sequences through internal amino acid modifications and conjugating them with natural amino acid side chains, along with incorporating an affinity group (biotin), facilitated the assembly of tetravalent bispecifics on a protein nanoplatform [69].

In brief, peptides containing pocket-binding domains have been designed to target the deep pocket and adjacent subpocket sites on the HIV-1 gp41 protein, which are essential for the fusion process. This approach highlights the importance of targeting both the deep cavity and subpocket sites on gp41 to inhibit HIV-1 infection effectively. Coreceptor binding site inhibitors, such as the tyrosine-sulfated peptides pV2α-Tys and pCCR5-Tys, have been identified as effective in mimicking the CCR5 N-terminus, thus blocking HIV-1 infection. Despite their potential, these sulfated V2 mimics have not significantly inhibited HIV-1 entry and fusion, which suggests that additional factors, such as the highly conserved C-terminal sulfotyrosine (Tys177), play a critical role in their effectiveness. The broader implication of these findings is the importance of the structural conservation of the gp120 coreceptor-binding site in developing effective inhibitors.

Research into the V3 loop of gp120 has led to the development of cyclic peptides with enhanced stability. Studies of these peptides have shown that they bind selectively to CXCR4, and this binding is maintained even with different chiralities of the peptides. This suggests that the V3 loop-CXCR4 interface can accommodate various ligand conformations, which might help the virus evade inhibition by maintaining coreceptor recognition despite mutations.

The development of innovative peptide constructs, such as the combination of VIRIP102C9 and EPI-X4 JM#173, has shown promise. VIRIP102C9 targets the gp41 fusion peptide, while EPI-X4 JM#173, an optimized CXCR4 antagonist, blocks the CXCR4 co-receptor. Integrating these peptides into a single construct has demonstrated enhanced activity against both CCR5- and CXCR4-tropic HIV-1 variants. This approach, which combines different thiol-reactive moieties and uses a protein nanoplatform, highlights the potential for creating multifunctional peptide-based therapies.

3.1.1.5. Irreversible Env Inactivators

Peptide triazoles (PTs) are a class of HIV-1 entry inhibitors that irreversibly inactivate Env trimers by exploiting the protein's inherent metastable nature. A related group, peptide triazole thiols (PTTs), can simultaneously bind to both the CD4 receptor and coreceptor sites on the Env gp120 subunit. This dual binding induces significant conformational changes in Env, enhancing their effectiveness as inhibitors [13]. Macrocyclic peptide triazoles (cPTs) are capable of binding to HIV-1 Env gp120 with nanomolar potency, leading to irreversible inactivation of the virus. The cyclic peptide triazoles AAR029b and FITC-AAR029b, as shown in Figure 9, demonstrated antiviral activity against HIV-1 with EC₅₀ values of 210 ± 10 nM and 320 ± 20 nM, respectively. FITC-AAR029b and AAR029b encapsulated in liposome formulations remained intact after 24 h of exposure to a human serum protease mixture while the corresponding linear peptide UM15 was rapidly degraded within minutes. These findings indicate that both AAR029b and its FITC-conjugated form maintained metabolic stability under physiological assay conditions [70]. Overall, PTs, exploited the metastable nature of Env trimers, and PTTs, which bind to both CD4 receptor and coreceptor sites on the gp120 subunit, enhance inhibition through significant conformational changes in Env.

3.1.1.6. Inhibitors Targeting gp41 Fusion Peptide (FP) Interactions

Fusion peptides (FPs) are crucial for the infection process of many viral pathogens, as they are inserted into the cellular membrane to facilitate entry. Initially, it was believed that FPs were difficult to target for therapy due to their limited accessibility. However, an optimized derivative of an endogenous α1-antitrypsin fragment, known as virus inhibitory peptide (VIRIP) (VIR-576), has shown promise by targeting the gp41 FP and effectively reducing viral loads in individuals infected with HIV-1. Münch et al (2007) indicated that FP inhibitors VIR-353 and VIR-576 significantly inhibited HIV-1 spread in ex vivo infected HLAC cultures, showing a notable reduction in viral activity at 100 nM and complete suppression of viral replication at 1 μM [71]. VIR-576, demonstrated effectiveness in a phase I/II clinical trial with a high genetic barrier to resistance. Despite this, partially resistant CXCR4-tropic HIV-1 variants emerged after extended passaging in the presence of another derivative, VIR-353, with seven mutations identified across the envelope glycoprotein but not within the gp41 fusion peptide. This indicates that the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is high [72].

A limited number of peptides have been established as fusion inhibitors that are not derived from gp41 domains. GB virus C (GBV-C) is a lymphotropic virus that infects humans and replicates in primary T and B lymphocytes. The 18-mer synthetic peptides EIP47 (WILEYLWKVPFDFWRGVI), E1P47-1 (ILEYLWKVPFDFWRGVIS), and E1P47-2 (LEYLWKVPFDFWRGVISL), derived from the GBV-C E1 protein (E1(139-156)), demonstrated antiviral activity against HIV-1 with IC₅₀ values of 2.7, 5.4 and 6.6 µM, respectively, and targeted the gp41 FP [73]. The E1P47 peptide interacts with the highly conserved N-terminal region of HIV-1 gp41 (the FP), crucial for viral entry, in a manner similar to the inhibitor VIR576.

Overall, the findings emphasize the potential of peptide-based inhibitors targeting the gp41 fusion peptide and incorporating innovative structural modifications, in advancing HIV-1 therapy.

3.1.1.7. Transmembrane Protein Sequence-Derived Anti-HIV Peptides

The reconstruction of transmembrane protein sequences from HIV-2 and simian immunodeficiency virus (SIV) led to the development of ancestral peptides derived from helical region 2 (HR2). Among these, the peptide P3 (WQEWEQQVRYFLEANISQRLEQAQIQQEKNMYELQKLNSWDVFGNWF) exhibited notable anti-HIV-1 activity, including against T-20-resistant variants, as well as against HIV-2, with low antigenicity and high stability. P3's ability to form a typical α-helical structure in solution and its strong binding to the transmembrane protein resulted in potent inhibition of both HIV-2 (mean IC₅₀ of 63.8 nM) and HIV-1 (mean IC₅₀ of 11 nM), including T-20-resistant isolates. The presence of the N43K mutation in the HR1 region of HIV-1 increased P3 resistance, indicating that P3 targets the HR1 region in the transmembrane glycoprotein [74].

4. Conclusions

5. Future Perspectives

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