Dual activity of amphiphilic Zn(II) nitroporphyrin derivatives as HIV-1 entry inhibitors and in cancer photodynamic therapy.

Two Zn(II) nitro porphyrin derivatives bearing combinations of meso-4-nitrophenyl and meso-4-methylpyridinium moieties and their free-base precursors were synthesized through one-pot microwave process, purified and characterized. The biological activity of these nitroporphyrins was assessed under both photodynamic and non-photodynamic conditions to correlate their structure-activity relationship (SAR). Unlike, the free-base precursors, Zn(II) complexes of these nitroporphyrins displayed nearly complete inhibition in the entry of lentiviruses such as HIV-1 and SIVmac under non-photodynamic conditions. In addition, the Zn(II) complexes also exhibited a higher in vitro photodynamic activity towards human lung cancer cell-line A549 than their free-base precursors. Our results strongly suggest that incorporation of Zn(II) has improved the antiviral and anticancer properties of the nitroporphyrins. To the best of our knowledge, this is the first report demonstrating the dual activity of nitroporphyrin-zinc complexes as antiviral and anti-cancer, which will aid in their development as therapeutics in clinics.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors.

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.

A novel multivalent DNA helix-based inhibitor showed enhanced anti-HIV-1 fusion activity.

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates, but further research is required to enhance their efficiency. The trimeric structure of the HIV-1 envelope glycoprotein provides a structural basis for multivalent drug design. In this work, a "multi-domain" strategy was adopted for design of an oligodeoxynucleotide with assembly, linkage, and activity domains. Built on the self-assembly of higher-order nucleic acid structure, a novel category of multivalent DNA helix-based HIV-1 fusion inhibitor could be easily obtained by a simple annealing course in solution buffer, with no other chemical synthesis for multivalent connection. An optimized multivalent molecule, M4, showed significantly higher anti-HIV-1 fusion activity than did corresponding monovalent inhibitors. Examination of the underlying mechanism indicated that M4 could interact with HIV-1 glycoproteins gp120 and gp41, thereby inhibiting 6HB formation in the fusion course. M4 also showed anti-RDDP and anti-RNase H activity of reverse transcriptase. Besides, these assembled molecules showed improved in vitro metabolic stability in liver homogenate, kidney homogenate, and rat plasma. Moreover, little acute toxicity was observed. Our findings aid in the structural design and understanding of the mechanisms of DNA helix-based HIV-1 inhibitors. This study also provides a general strategy based on a new structural paradigm for the design of other multivalent nucleic acid drugs.

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer.

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett.2015, 25 2853-59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC50=37.81µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.

Small molecule inhibitors of HIVgp41 N-heptad repeat trimer formation.

Identification of mechanistically novel anti-HIV fusion inhibitors was accomplished using a computer-aided structure-based design approach with the goal of blocking the formation of the N-heptad repeat (NHR) trimer of the viral protein gp41. A virtual screening strategy that included per-residue interaction patterns (footprints) was employed to identify small molecules compatible with putative binding pockets at the internal interface of the NHR helices at the core native viral six-helix bundle. From a screen of ∼2.8 million compounds using the DOCK program, 120 with favorable energetic and footprint overlap characteristics were purchased and experimentally tested leading to two compounds with favorable cell-cell fusion (IC50) and cytotoxicity profiles. Importantly, both hits were identified on the basis of scores containing footprint overlap terms and would not have been identified using the standard DOCK energy function alone. To our knowledge, these compounds represent the first reported small molecules that inhibit viral entry via the proposed NHR-trimer obstruction mechanism.

Evaluation of potential genotoxicity of HIV entry inhibitors derived from natural sources.

AIDS is a global pandemic that has seen the development of novel and effective treatments to improve the quality of life of those infected and reduction of spread of the disease. Palmitic Acid (PA), which we identified and isolated from Sargassum fusiforme, is a naturally occurring fatty acid that specifically inhibits HIV entry by binding to a novel pocket on the CD4 receptor. We also identified a structural analogue, 2-bromopalmitate (2-BP), as a more effective HIV entry inhibitor with a 20-fold increase in efficacy. We have used the structure-activity relationship (SAR) of 2-BP as a platform to identify new small chemical molecules that fit into the various identified active sites in an effort to identify more potent CD4 entry inhibitors. To validate further drug development, we tested the PA and 2-BP scaffold molecules for genotoxic potential. The FDA and International Conference on Harmonisation (ICH) recommends using a standardized 3-test battery for testing compound genotoxicity consisting of the bacterial reverse mutation assay, mouse lymphoma assay, and rat micronucleus assay. PA and 2-BP and their metabolites tested negative in all three genotoxicty tests. 2-BP is the first derivative of PA to undergo pre-clinical screening, which will enable us to now test multiple simultaneous small chemical structures based on activity in scaffold modeling across the dimension of pre-clinical testing to enable transition to human testing.

Identification of a human protein-derived HIV-1 fusion inhibitor targeting the gp41 fusion core structure.

The HIV-1 envelope glycoprotein (Env) gp41 plays a crucial role in the viral fusion process. The peptides derived from the C-terminal heptad repeat (CHR) of gp41 are potent HIV fusion inhibitors. However, the activity of these anti-HIV-1 peptides in vivo may be attenuated by their induction of anti-gp41 antibodies. Thus, it is essential to identify antiviral peptides or proteins with low, or no, immunogenicity to humans. Here, we found that the C-terminal fragment (aa 462-521) of the human POB1 (the partner of RalBP1), designated C60, is an HIV-1 fusion inhibitor. It bound to N36, the peptide derived from the N-terminal heptad repeat (NHR) of gp41, and to the six-helix bundle (6-HB) formed by N36 and C34, a CHR-peptide, but it did not bind to C34. Unlike the CHR-peptides, C60 did not block gp41 6-HB formation. Rather, results suggest that C60 inhibits HIV-1 fusion by binding to the 6-HB, in particular, the residues in the gp41 NHR domain that are exposed on the surface of 6-HB. Since 6-HB plays a crucial role in the late stage of fusion between the viral envelope and endosomal membrane during the endocytic process of HIV-1, C60 may serve as a host restriction factor to suppress HIV-1 entry into CD4+ T lymphocytes. Taken together, it can be concluded from these results that C60 can be used as a lead for the development of anti-HIV-1 therapeutics or microbicides for the treatment and prevention of HIV-1 infection, as well as a molecular probe to study the fusogenic mechanism of HIV-1.

Amino acid derivatives of the (-) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1.

T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (-) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (-) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (-) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (-) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(-) gossypol derivative ((-)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (-)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (-)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (-)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (-) gossypol and amino acid derivatives of (-) gossypol are also discussed. Collectively, our results indicate that (-)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.

In situ forming implant for controlled delivery of an anti-HIV fusion inhibitor.

An injectable, phase sensitive, in situ forming, implantable delivery system was developed for enfuvirtide, a therapeutic peptide used in the treatment of HIV infection. The development studies were carried out using poly (D,L-lactide-co-glycolide), a smart, biodegradable polymer. Different formulations were designed, prepared and evaluated by employing response surface, optimal design of experiment technique. The optimized formulation was identified and validated for its performance by using numerical optimization technique. The in vitro evaluation parameters included rheology, compatibility studies, drug release as well as conformational and physicochemical stability studies. In vivo pharmacokinetic parameters and biocompatibility studies were determined in rat models and were statistically analyzed. It was found that the optimized formulation extended the enfuvirtide release and maintained the drug plasma concentration within therapeutically effective range up to 48 h. The optimized formulation maintained physicochemical and conformational stability for at least 6 months and was biocompatible with the animal tissue.

Antiviral breadth and combination potential of peptide triazole HIV-1 entry inhibitors.

The first stage of human immunodeficiency virus type 1 (HIV-1) infection involves the fusion of viral and host cellular membranes mediated by viral envelope glycoprotein gp120. Inhibitors that specifically target gp120 are gaining increased attention as therapeutics or preventatives to prevent the spread of HIV-1. One promising new group of inhibitors is the peptide triazoles, which bind to gp120 and simultaneously block its interaction with both CD4 and the coreceptor. In this study, we assessed the most potent peptide triazole, HNG-156, for inhibitory breadth, cytotoxicity, and efficacy, both alone and in combination with other antiviral compounds, against HIV-1. HNG-156 inhibited a panel of 16 subtype B and C isolates of HIV-1 in a single-round infection assay. Inhibition of cell infection by replication-competent clinical isolates of HIV-1 was also observed with HNG-156. We found that HNG-156 had a greater than predicted effect when combined with several other entry inhibitors or the reverse transcriptase inhibitor tenofovir. Overall, we find that HNG-156 is noncytotoxic, has a broad inhibition profile, and provides a positive combination with several inhibitors of the HIV-1 life cycle. These results support the pursuit of efficacy and toxicity analyses in more advanced cell and animal models to develop peptide triazole family inhibitors of HIV-1 into antagonists of HIV-1 infection.

Biophysical investigations of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide.

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research.

A RhoA-derived peptide inhibits human immunodeficiency virus-1 entry in vitro.

RhoA-derived peptides have been shown to have antiviral activity against both human respiratory syncytial virus and human parainfluenza virus-3. The present study investigates the toxicity, anti-HIV-1 activity and mechanism of action of a RhoA-derived peptide (RhoA 77-95). The efficacy of this peptide was compared to a scrambled peptide of the same amino acid composition and Enfuvirtide, a HIV entry inhibitor. Our data show that this RhoA-derived peptide is a non-toxic and effective inhibitor of a CXCR4 tropic strain of HIV-1. We also demonstrate that the mechanism of entry inhibition is likely mediated by polyanionic properties and is dependent on the dimerization of peptides.

CD4 mimics targeting the HIV entry mechanism and their hybrid molecules with a CXCR4 antagonist.

Small molecules behaving as CD4 mimics were previously reported as HIV-1 entry inhibitors that block the gp120-CD4 interaction and induce a conformational change in gp120, exposing its co-receptor-binding site. A structure-activity relationship (SAR) study of a series of CD4 mimic analogs was conducted to investigate the contribution from the piperidine moiety of CD4 mimic 1 to anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. In addition, several hybrid molecules based on conjugation of a CD4 mimic analog with a selective CXCR4 antagonist were also synthesized and their utility evaluated.

Sifuvirtide, a potent HIV fusion inhibitor peptide.

Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC(50)), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC(50)) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1(IIIB) were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

In vitro pre- and post-exposure prophylaxis using HIV inhibitors as microbicides against cell-free or cell-associated HIV-1 infection.

Several classes of microbicides are being evaluated for the prevention of sexual HIV transmission. In vivo, the infectious dose and viral source involved in transmission remain uncertain and it is likely that women will use microbicides both before and after high-risk HIV exposure. Therefore, we evaluated HIV entry inhibitors (EIs) and reverse transcriptase inhibitors (RTIs) for their ability to block cell-free and cell-associated HIV-1 infection in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4+ T-cells using settings of pre- and post-exposure prophylaxis. In the pre-exposure assay, where compound was present before, during and 24 h after infection, all tested EIs (BMS806, TAK779 and T20) and RTIs (PMPA, TMC120 and UC781) blocked infection with 10(-4) multiplicity of infection (MOI) of cell-free virus at a dose between 100 and 10,000 nM, dependent on the compound used. At 10(-3) MOI, however, only T20 and the RTIs completely blocked infection. Furthermore, in experiments with cell-associated virus, EIs were ineffective, whereas RTIs actively blocked infection with similar potency as in the experiments with cell-free virus. In the post-exposure assay, where compound was added 2 h after infection and remained present for 24 h, EIs were inactive whereas RTIs blocked cell-free and cell-associated viral infections equally efficiently. Moreover, post-exposure prophylaxis initiated 24 h after infection with cell-free or cell-associated HIV-1 was still effective with 1,000 nM of TMC120. Both EIs and RTIs were non-cytotoxic at any tested concentration for MO-DC and CD4+ T-cells in co-culture. Our study shows that RTIs are potent inhibitors of cell-free and cell-associated virus used either in pre- or post-exposure settings. It highlights that parameters such as viral input, viral source, the time of compound addition and the target cells should be considered in microbicides evaluation.

N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion.

A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs. However, T-20 must be injected twice daily and is too expensive. Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors. By screening a chemical library consisting of "drug-like" compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range. The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity. NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the alpha-helical conformation. They blocked a d-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain. Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge. These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41.

Safety, tolerability, and plasma pharmacokinetics of high-strength formulations of enfuvirtide (T-20) in treatment-experienced HIV-1-infected patients.

BACKGROUND: Enfuvirtide, a HIV-1 membrane fusion inhibitor, is the first viral entry inhibitor approved for the treatment of HIV-1 infected patients in the USA. Parenteral administration of enfuvirtide in clinical trials has been safe and has resulted in significant decreases in plasma viral load, even in the setting of extensive previous treatment and multi-drug resistance to conventional antiretroviral (ARV) therapy. Previous formulations have required two injections administered twice-daily (BID). OBJECTIVES: The primary objectives of this study were to evaluate the safety, tolerability, and pharmacokinetics of two high-strength 100 mg/ml formulations of enfuvirtide (carbonate [CO(3)] and tromethamine [TRIS] buffer) and of the current formulation (50 mg/ml CO(3) formulation) at doses of 90 mg (deliverable) BID and 67.5 mg (deliverable) BID in treatment-experienced patients. STUDY DESIGN: This was a phase II, multi-center, open-label, sequential cross-over pharmacokinetic, efficacy, and safety study. Study design included two treatment variables; dose (90 mg or 67.5 mg BID) and formulation (A: 50 mg/ml CO(3), B: 100 mg/ml CO(3) or C; 100 mg/ml TRIS). RESULTS: Forty-six treatment-experienced participants were sequentially enrolled into three treatment cohorts. All cohorts had similar safety profiles and only one patient discontinued due to an adverse event. Pharmacokinetic data indicated that the high-strength 100 mg/ml CO(3) formulation was bioequivalent to the 50 mg/ml CO(3) formulation whereas the TRIS formulation was not. At 48 weeks, 59.1%, 66.7% and 16.7% had <400 copies per milliliter HIV-1 RNA in the 90 MgCO(3), 67.5 MgCO(3) and 90 mg TRIS cohorts with median suppression of HIV-1 RNA of 2.97, 3.48, and 0.87 log(10)copies per milliliter, respectively. CONCLUSIONS: Based upon bioequivalence data and the convenience and similarity in safety and virological effect with the 50 mg/ml formulation, the 100 mg/ml CO(3) formulation was selected for use in clinical efficacy studies of enfuvirtide.