Characterization of a CXCR4 antagonist TIQ-15 with dual tropic HIV entry inhibition properties.

The chemokine co-receptors CXCR4 and CCR5 mediate HIV entry and signal transduction necessary for viral infection. However, to date only the CCR5 antagonist maraviroc is approved for treating HIV-1 infection. Given that approximately 50% of late-stage HIV patients also develop CXCR4-tropic virus, clinical anti-HIV CXCR4 antagonists are needed. Here, we describe a novel allosteric CXCR4 antagonist TIQ-15 which inhibits CXCR4-tropic HIV-1 infection of primary and transformed CD4 T cells. TIQ-15 blocks HIV entry with an IC50 of 13 nM. TIQ-15 also inhibits SDF-1α/CXCR4-mediated cAMP production, cofilin activation, and chemotactic signaling. In addition, TIQ-15 induces CXCR4 receptor internalization without affecting the levels of the CD4 receptor, suggesting that TIQ-15 may act through a novel allosteric site on CXCR4 for blocking HIV entry. Furthermore, TIQ-15 did not inhibit VSV-G pseudotyped HIV-1 infection, demonstrating its specificity in blocking CXCR4-tropic virus entry, but not CXCR4-independent endocytosis or post-entry steps. When tested against a panel of clinical isolates, TIQ-15 showed potent inhibition against CXCR4-tropic and dual-tropic viruses, and moderate inhibition against CCR5-tropic isolates. This observation was followed by a co-dosing study with maraviroc, and TIQ-15 demonstrated synergistic activity. In summary, here we describe a novel HIV-1 entry inhibitor, TIQ-15, which potently inhibits CXCR4-tropic viruses while possessing low-level synergistic activities against CCR5-tropic viruses. TIQ-15 could potentially be co-dosed with the CCR5 inhibitor maraviroc to block viruses of mixed tropisms.

Design of Artificial C-Peptides as Potential Anti-HIV-1 Inhibitors Based on 6-HB Formation Mechanism.

BACKGROUND: The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors. OBJECTIVE: The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction. METHODS: The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software. RESULTS: The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation. CONCLUSION: We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

In vitro phenotypic susceptibility of HIV-1 non-group M to CCR5 inhibitor (maraviroc): TROPI-CO study.

The susceptibility of genetically divergent HIV-1 strains (HIV-1 non-M) from groups O, N, and P to the CCR5 co-receptor antagonist, maraviroc (MVC), was investigated among a large panel of 45 clinical strains, representative of the viral genetic diversity. The results were compared to the reference strains of HIV-1 group M (HIV-1/M) with known tropism. Among the non-M strains, a wide range of phenotypic susceptibilities to MVC were observed. The large majority of HIV-1/O strains (40/42) displayed a high susceptibility to MVC, with median and mean IC50 values of 1.23 and 1.33 nM, respectively, similar to the HIV-1/M R5 strain (1.89 nM). However, the two remaining HIV-1/O strains exhibited a lower susceptibility (IC50 at 482 and 496 nM), in accordance with their dual/mixed (DM) tropism. Interestingly, the two HIV-1/N strains demonstrated varying susceptibility patterns, despite always having relatively low IC50 values (2.87 and 47.5 nM). This emphasized the complexity of determining susceptibility solely based on IC50 values. Our study examined the susceptibility of all HIV-1 non-M groups to MVC and correlated these findings with virus tropism (X4, R5, or DM). The results confirm the critical significance of tropism determination before initiating MVC treatment in patients infected with HIV-1 non-M. Furthermore, we advocate for the consideration of additional parameters, such as the slope of inhibition curves, to provide a more thorough characterization of phenotypic susceptibility profiles. IMPORTANCE: Unlike HIV-1 group M, the scarcity of studies on HIV-1 non-M groups (O, N, and P) presents challenges in understanding their susceptibility to antiretroviral treatments, particularly due to their natural resistance to non-nucleoside reverse transcriptase inhibitors. The TROPI-CO study logically complements our prior investigations into integrase inhibitors and anti-gp120 efficacy. The largest panel of 45 non-M strains existing so far yielded valuable results on maraviroc (MVC) susceptibility. The significant variations in MVC IC50 reveal a spectrum of susceptibilities, with most strains displaying R5 tropism. Notably, the absence of MVC-resistant strains suggests a potential therapeutic avenue. The study also employs a robust novel cell-based phenotropism assay and identifies distinct groups of susceptibilities based on inhibition curve slopes. Our findings emphasize the importance of determining tropism before initiating MVC and provide crucial insights for selecting effective therapeutic strategies in the delicate context of HIV-1 non-M infections.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

Frequency of Fusion Inhibitor Resistance Mutations Among Therapy-Naïve HIV Patients.

Glycoprotein 41 (gp41) of the human immunodeficiency virus type 1 (HIV-1) protein plays a critical role in membrane fusion. Gp41 binds to proteins in the plasma membrane of CD4+ T cells, particularly the T-cell antigen receptor (TCR). These findings indicate that gp41 is involved in the assembly of HIV-1 at the plasma membrane of T cells and affects the stimulation of the TCR. To control HIV-1, new inhibitors were introduced to target the gp41 protein. However, mutations in this region might reduce their efficacy. The Gp41 region was amplified from the sera of 30 patients using nested polymerase chain reaction. The sequences were analyzed by bioinformatics tools to identify mutations and gp41 structural features. Subtyping and the interaction between fusion inhibitors and gp41 proteins were also examined. As the first report from Iran, docking analysis between fusion inhibitors and Iranian gp41 proteins showed that mutations in gp41 could not reduce the efficacy of the fusion inhibitors. Most of the patients were infected with CRF35-AD. Several post-modification positions, including glycosylation and phosphorylation sites, were identified in the gp41 protein. Our findings revealed no known multinational drug resistance to gp41 inhibitors; thus, fusion inhibitors can effectively inhibit HIV in Iranian patients. In addition, the present study introduced a new gp41 region (36-44 aa), which considerably influences the interactions between gp41 inhibitors and the gp41 protein. This region may play a pivotal role in suppressing gp41 inhibitors in CFR35-AD. Furthermore, gp41 can be considered a good target for subtyping analysis via the phylogenetic method.

Improving Anti-HIV activity and pharmacokinetics of enfuvirtide (T20) by modification with oligomannose.

Dendritic cells (DCs) play a pivotal role in controlling HIV-1 infections of CD4+ T cells. DC-SIGN, which is expressed on the surface of DCs, efficiently captures HIV-1 virions by binding to the highly mannosylated membrane protein, gp120, and then the DCs transport the virus to target T cells in lymphoid organs. This study explored the modification of T20, a peptide inhibitor of HIV-1 fusion, by conjugation of the N-terminus with varying sizes of oligomannose, which are DC-SIGN-specific carbohydrates, aiming to create dual-targeting HIV inhibitors. Mechanistic studies indicated the dual-target binding of the conjugates. Antiviral assays demonstrated that N-terminal mannosylation of T20 resulted in increased inhibition of the viral infection of TZM-b1 cells (EC50 = 0.3-0.8 vs. 1.4 nM). Pentamannosylated T20 (M5-T20) exhibited a stronger inhibitory effect on virus entry into DC-SIGN+ 293T cells compared with T20 (67% vs. 50% inhibition at 500 µM). M5-T20 displayed an extended half-life in rats relative to T20 (T1/2: 8.56 vs. 1.64 h, respectively). These conjugates represent a potential new treatment for HIV infections with improved antiviral activity and pharmacokinetics, and this strategy may prove useful in developing dual-target inhibitors for other pathogens that require DC-SIGN involvement for infection.

Natural products as a source of Coronavirus entry inhibitors.

The COVID-19 pandemic has had a significant and lasting impact on the world. Four years on, despite the existence of effective vaccines, the continuous emergence of new SARS-CoV-2 variants remains a challenge for long-term immunity. Additionally, there remain few purpose-built antivirals to protect individuals at risk of severe disease in the event of future coronavirus outbreaks. A promising mechanism of action for novel coronavirus antivirals is the inhibition of viral entry. To facilitate entry, the coronavirus spike glycoprotein interacts with angiotensin converting enzyme 2 (ACE2) on respiratory epithelial cells. Blocking this interaction and consequently viral replication may be an effective strategy for treating infection, however further research is needed to better characterize candidate molecules with antiviral activity before progressing to animal studies and clinical trials. In general, antiviral drugs are developed from purely synthetic compounds or synthetic derivatives of natural products such as plant secondary metabolites. While the former is often favored due to the higher specificity afforded by rational drug design, natural products offer several unique advantages that make them worthy of further study including diverse bioactivity and the ability to work synergistically with other drugs. Accordingly, there has recently been a renewed interest in natural product-derived antivirals in the wake of the COVID-19 pandemic. This review provides a summary of recent research into coronavirus entry inhibitors, with a focus on natural compounds derived from plants, honey, and marine sponges.

Side-by-side comparison of published small molecule inhibitors against thapsigargin-induced store-operated Ca2+ entry in HEK293 cells.

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.

Cross-resistance to entry inhibitors and lenacapavir resistance through Week 52 in study CAPELLA.

BACKGROUND: Lenacapavir (LEN) is a first-in-class inhibitor of human immunodeficiency virus type 1 (HIV-1) capsid function for the treatment of heavily treatment-experienced people with HIV (PWH) harbouring multidrug resistance in combination with an optimized background regimen (OBR). Here, we describe in vitro analysis of the interplay between entry inhibitors (EI; enfuvirtide, fostemsavir, ibalizumab, and maraviroc) susceptibility and LEN susceptibility in samples from 72 participants in the phase 2/3 CAPELLA study, as well as the emergence of resistance in CAPELLA through 52 weeks. METHODS: The phenotypic susceptibility to EIs of screening samples from participants was analysed using entry assays, and susceptibility to LEN was generated. Genotypic and phenotypic resistance to LEN was evaluated for subjects with virological failure through Week 52. RESULTS: Overall, viruses with resistance to EIs showed no cross-resistance to LEN, with a mean fold change from wild type close to 1.0. Of the 22 participants analysed for resistance through Week 52, 9 participants (13%) had emergence of capsid resistance mutation(s) while the remaining 13 participants (18%) had no change in the capsid sequence. CONCLUSION: The gag sequence from EI-resistant isolates did not affect LEN susceptibility. The lack of cross-resistance to LEN across ARV-resistant isolates supports the use of LEN in PWH regardless of their treatment history. During the second half-year period of the CAPELLA Study, development of LEN resistance was rare and was overall associated with functional LEN monotherapy due to either nonadherence or resistance-driven non-susceptibility to OBR.

Synthetic approaches and application of clinically approved small-molecule Anti-HIV drugs: An update.

Application of chemotherapeutic agents to inhibit the HIV replication process has brought about a significant metamorphosis in the landscape of AIDS. Substantial declines in morbidity and mortality rates have been attained, accompanied by notable decreases in healthcare resource utilization. However, treatment modalities do not uniformly inhibit HIV replication in every patient, while the emergence of drug-resistant viral strains poses a substantial obstacle to subsequent therapeutic interventions. Furthermore, chronic administration of therapy may lead to the manifestation of toxicities. These challenges necessitate the exploration of novel pharmacological agents and innovative therapeutic approaches aimed at effectively managing the persistent viral replication characteristic of chronic infection. This review examines the role of clinically approved small-molecule drugs in the treatment of HIV/AIDS, which provides an in-depth analysis of the major classes of small-molecule drugs, including nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), integrase inhibitors, entry inhibitors, and pharmacokinetic enhancers. The review mainly discusses the application, synthetic routes, and mechanisms of action of small-molecule drugs employed in the treatment of HIV, as well as their use in combination with antiretroviral therapy, presenting viewpoints on forthcoming avenues in the development of novel anti-HIV drugs.

Structure-function analyses reveal key molecular determinants of HIV-1 CRF01_AE resistance to the entry inhibitor temsavir.

The HIV-1 entry inhibitor temsavir prevents the viral receptor CD4 (cluster of differentiation 4) from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this, temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveals that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad antiviral activity.

SELEX based aptamers with diagnostic and entry inhibitor therapeutic potential for SARS-CoV-2.

Frequent mutation and variable immunological protection against vaccination is a common feature for COVID-19 pandemic. Early detection and confinement remain key to controlling further spread of infection. In response, we have developed an aptamer-based system that possesses both diagnostic and therapeutic potential towards the virus. A random aptamer library (~ 1017 molecules) was screened using systematic evolution of ligands by exponential enrichment (SELEX) and aptamer R was identified as a potent binder for the SARS-CoV-2 spike receptor binding domain (RBD) using in vitro binding assay. Using a pseudotyped viral entry assay we have shown that aptamer R specifically inhibited the entry of a SARS-CoV-2 pseudotyped virus in HEK293T-ACE2 cells but did not inhibit the entry of a Vesicular Stomatitis Virus (VSV) glycoprotein (G) pseudotyped virus, hence establishing its specificity towards SARS-CoV-2 spike protein. The antiviral potential of aptamers R and J (same central sequence as R but lacking flanked primer regions) was tested and showed 95.4% and 82.5% inhibition, respectively, against the SARS-CoV-2 virus. Finally, intermolecular interactions between the aptamers and the RBD domain were analyzed using in silico docking and molecular dynamics simulations that provided additional insight into the binding and inhibitory action of aptamers R and J.

Novel mono- and multivalent N-acetylneuraminic acid glycoclusters as potential broad-spectrum entry inhibitors for influenza and coronavirus infection.

N-acetylneuraminic acid (Neu5Ac) is a glycan receptor of viruses spread in many eukaryotic cells. The present work aimed to design, synthesis and biological evaluation of a panel of Neu5Ac derivatives based on a cyclodextrin (CD) scaffold for targeting influenza and coronavirus membrane proteins. The multivalent Neu5Ac glycoclusters efficiently inhibited chicken erythrocyte agglutination induced by intact influenza virus in a Neu5Ac density-dependent fashion. Compared with inhibition by Neu5Ac, the multivalent inhibitor with 21 Neu5Ac residues on the primary face of the ß-CD scaffold afforded 1788-fold higher binding affinity inhibition for influenza virus hemagglutinin with a dissociation constant (KD) of 3.87 × 10-7 M. It showed moderate binding affinity to influenza virus neuraminidase, but with only about one-thirtieth the potency of that with the HA protein. It also exhibited strong binding affinity to the spike protein of three human coronaviruses (severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and severe acute respiratory syndrome coronavirus 2), with KD values in the low micromolar range, which is about 10-time weaker than that of HA. Therefore, these multivalent sialylated CD derivatives have possible therapeutic application as broad-spectrum antiviral entry inhibitors for many viruses by targeting the Neu5Ac of host cells.

Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides.

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.

A dePEGylated Lipopeptide-Based Pan-Coronavirus Fusion Inhibitor Exhibits Potent and Broad-Spectrum Anti-HIV-1 Activity without Eliciting Anti-PEG Antibodies.

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses.

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities.

Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 µM while the standard one had 0.0180 µM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.

Broad-spectrum anti-HIV activity and high drug resistance barrier of lipopeptide HIV fusion inhibitor LP-19.

Lipopeptide-19, a HIV fusion inhibitor (LP-19), has showed potent anti-HIV activity. However, there is still limited information of the antiviral activity against different subtype clinical isolates and the drug resistance barrier of LP-19. Therefore, 47 HIV clinical isolates were selected for this study. The viral features were identified, in which 43 strains are CCR5 tropisms, and 4 strains are CCR5/CXCR4 tropisms, and there are 6 subtype B', 15 CRF01_AE, 14 CRF07_BC, 2 CRF08_BC and 10 URF strains. These 47 viruses were used to detected and analyze the inhibitory activities of LP-19. The results showed that the average 50% inhibitory concentration (IC50) and 90% inhibitory concentration (IC90) of LP-19 were 0.50 nM and 1.88 nM, respectively. The average IC50 of LP-19 to B', CRF01_AE, CRF07_BC, CRF08_BC, and URF strains was 0.76 nM, 0.29 nM, 0.38 nM, 0.85 nM, and 0.44 nM, respectively. C34 and Enfuvirtide (T-20), two fusion inhibitors, were compared on the corresponding strains simultaneously. The antiviral activity of LP-19 was 16.7-fold and 86-fold higher than that of C34 and T-20. The antiviral activity of LP-19, C34, and T-20 were further detected and showed IC50 was 0.15 nM, 1.02 nM, and 66.19 nM, respectively. IC50 of LP-19 was about 7-fold and 441-fold higher compared to C34 and T-20 against HIV-1 NL4-3 strains. NL4-3 strains were exposed to increasing concentrations of LP-19 and C34 in MT-2 cell culture. The culture virus was sequenced and analyzed. The results showed that A243V mutation site identified at weeks 28, 32, 38, and 39 of the cell culture in the gp41 CP (cytoplasmic domain) region. NL4-3/A243V viruses containing A243V mutation were constructed. Comparing the antiviral activities of LP-19 against HIV NL4-3 to HIV strains (only 1.3-fold), HIV did not show drug resistance when LP-19 reached 512-fold of the initial concentration under the drug pressure for 39 weeks. This study suggests that LP-19 has broad-spectrum anti-HIV activity, and high drug resistance barrier.

An Artificial Peptide-Based Bifunctional HIV-1 Entry Inhibitor That Interferes with Viral Glycoprotein-41 Six-Helix Bundle Formation and Antagonizes CCR5 on the Host Cell Membrane.

Human immunodeficiency virus type 1 (HIV-1) is characterized by high variability and drug resistance. This has necessitated the development of antivirals with a new chemotype and therapy. We previously identified an artificial peptide with non-native protein sequence, AP3, with the potential to inhibit HIV-1 fusion through targeting hydrophobic grooves on the N-terminal heptad repeat trimer of viral glycoprotein gp41. Here, a small-molecule HIV-1 inhibitor targeting chemokine coreceptor CCR5 on the host cell was integrated into the AP3 peptide, producing a novel dual-target inhibitor with improved activity against multiple HIV-1 strains including those resistant to the currently used anti-HIV-1 drug enfuvirtide. Its superior antiviral potency in comparison with the respective pharmacophoric moieties is in consonance with the dual binding of viral gp41 and host factor CCR5. Therefore, our work provides a potent artificial peptide-based bifunctional HIV-1 entry inhibitor and highlights the multitarget-directed ligands approach in the development of novel therapeutic anti-HIV-1 agents.

Development of a HPLC fluorometric method for the quantification of enfuvirtide following in vitro releasing studies on thermosensitive in situ forming gel.

Due to the presence of peptidase and protease in the gastrointestinal tract, peptides are subjected to digestion and inactivation when administrated orally. To avoid degradation and maintain the desired efficacy of peptide drugs, there is a demand to develop transdermal and intradermal delivery systems. This requires efficient and specific analytical methods to separate and quantify the peptide drugs from the formulation and the skin matrix in the early stages of pharmaceutical development. A high-performance liquid chromatography (HPLC) system equipped with a fluorometric detector was used to quantify enfuvirtide, which is the first fusion inhibitor for HIV treatment. The HPLC method was developed and validated according to the ICH Q2(R1) guidelines. The viability of the method was demonstrated during in vitro studies, where samples were analysed following intradermal administration of a thermosensitive in situ forming gel. Compared with previously reported methods, this assay proved efficient, sensitive and accurate, with a detection limit of 0.74 µg/mL and a run time of 9 min, mitigating the use of any internal standards and detergents. The addition of an organic solvent to the samples successfully solved the problem of low recovery caused by the adsorption of the drug to the plastic consumables in the sample treatment process. The amount of enfuvirtide releasing from the in situ gel through skin after 7 hours was 16.25 ± 7.08 µg, which was significantly lower than the reconstituted FUZEON® itself (26.68 ± 10.45 µg), showing a longer release profile. The results may be beneficial as a constructive input for future enfuvirtide quantification within a preclinical setting through in vitro release studies across the skin.

Multitargeted drug design strategy for discovery of short-peptide-based HIV-1 entry inhibitors with high potency.

The development of short-peptide-based inhibitors to prevent HIV-1 entry into the host cell has been rewarded with limited success. Herein, we report a multitarget-directed ligand strategy to generate a series of short-peptide HIV-1 entry inhibitors that integrated the pharmacological activities of a peptide fusion inhibitor able to disrupt HIV-1 gp41 glycoprotein hexameric coiled-coil assembly and a small-molecule CCR5 antagonist that blocks the interaction between HIV-1 and its coreceptor. Among these inhibitors, dual-target 23-residue peptides SP12T and SP12L displayed dramatically increased inhibitory activities against HIV-1 replication as compared to the marketed 36-residue peptide T20. Moreover, results suggested that SP12T and SP12L successfully performed a dual-targeting mechanism. It can be concluded that these short-peptide-based HIV-1 entry inhibitors have potential for further development as candidates for a novel multitarget therapy to treat HIV-1 infection.