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In this study, 37 derivatives of phorbol esters were synthesized and their anti-HIV-1 activities evaluated, building upon our previous synthesis of 51 phorbol derivatives. 12-Para-electron-acceptor- trans -cinnamoyl-13-decanoyl phorbol derivatives stood out, demonstrating remarkable anti-HIV-1 activities and inhibitory effects on syncytia formation. These derivatives exhibited a higher safety index compared with the positive control drug. Among them, 12-(trans -4-fluorocinnamoyl)-13-decanoyl phorbol, designated as compound 3c , exhibited the most potent anti-HIV-1 activity (EC 50 2.9 nmol·L−1, CC 50 /EC 50 11 117.24) and significantly inhibited the formation of syncytium (EC 50 7.0 nmol·L−1, CC 50 /EC 50 4891.43). Moreover, compound 3c is hypothesized to act both as an HIV-1 entry inhibitor and as an HIV-1 reverse transcriptase inhibitor. Isothermal titration calorimetry and molecular docking studies indicated that compound 3c may also function as a natural activator of protein kinase C (PKC). Therefore, compound 3c emerges as a potential candidate for developing new anti-HIV drugs. [ABSTRACT FROM AUTHOR]

The combination of three or more antiviral agents that act on different targets is known as highly active antiretroviral therapy (HAART), which is widely used to control HIV infection. However, because drug resistance and adverse effects occur after long-term administration, an increasing number of HIV/AIDS patients do not tolerate HAART. It is necessary to continue developing novel anti-HIV drugs, particularly HIV entry/fusion inhibitors. Our group previously identified a small-molecule compound, NB-64, with weak anti-HIV activity. Here, we found that N-substituted pyrrole derivative 12m (NSPD-12m), which was derived from NB-64, had strong anti-HIV-1 activity, and NSPD-12m-treated cells showed good viability. The mechanism of action of NSPD-12m might be targeting the gp41 transmembrane subunit of the HIV envelope glycoprotein, thus inhibiting HIV entry. Site-directed mutagenesis confirmed that a positively charged lysine residue (K574) located in the gp41 pocket region is pivotal for the binding of NSPD-12m to gp41. These findings suggest that NSPD-12m can serve as a lead compound to develop novel virus entry inhibitors.

The combination of three or more antiviral agents that act on different targets is known as highly active antiretroviral therapy (HAART), which is widely used to control HIV infection. However, because drug resistance and adverse effects occur after long-term administration, an increasing number of HIV/AIDS patients do not tolerate HAART. It is necessary to continue developing novel anti-HIV drugs, particularly HIV entry/fusion inhibitors. Our group previously identified a small-molecule compound, NB-64, with weak anti-HIV activity. Here, we found that N-substituted pyrrole derivative 12m (NSPD-12m), which was derived from NB-64, had strong anti-HIV-1 activity, and NSPD-12m-treated cells showed good viability. The mechanism of action of NSPD-12m might be targeting the gp41 transmembrane subunit of the HIV envelope glycoprotein, thus inhibiting HIV entry. Site-directed mutagenesis confirmed that a positively charged lysine residue (K574) located in the gp41 pocket region is pivotal for the binding of NSPD-12m to gp41. These findings suggest that NSPD-12m can serve as a lead compound to develop novel virus entry inhibitors. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The feasibility of utilizing the cell surface chemokine receptor CXCR4 for human immunodeficiency virus (HIV) entry inhibition and as an intracellular portal for targeted drug delivery was evaluated. Novel DV3 ligands (1DV3, 2DV3, and 4DV3) were designed, synthesized and conjugated to various probes (fluorescein isothiocyanate (FITC) or biotin) and cargos with sizes ranging from 10 to 50 nm (polyethylene glycol (PEG), streptavidin, and a polymeric nanoparticle). 4DV3 conjugated probes inhibited HIV-1 entry into the CXCR4-expressing reporter cell line TZM-bl (IC 50 at 553 nM) whereas 1DV3 and 2DV3 did not. 4DV3 also inhibited binding of anti-CXCR4 antibody 44,708 to TZM-bl cells with nanomolar potency, while the small-molecule CXCR4 antagonist AMD3100 did not. Molecular modeling suggested simultaneous binding of a single 4DV3 molecule to four CXCR4 molecules. Differences in CXCR4-binding sites could explain the discrete inhibitory effects observed for 4DV3, the 44,708 antibody and AMD3100. In the Sup-T1 cell chemotaxis assay, the 4DV3 ligand functioned as a CXCR4 allosteric enhancer. In addition, 4DV3 ligand-conjugated cargos with sizes ranging from 10 to 50 nm were taken up into CXCR4-expressing Sup-T1 and TZM-bl cells, demonstrating that CXCR4 could serve as a drug delivery portal for nanocarriers. The uptake of 4DV3 functionalized nanocarriers combined with the allosteric interaction with CXCR4 suggests enhanced endocytosis occurs when 4DV3 is the targeting ligand. The current results indicate that 4DV3 might serve as a prototype for a new type of dual function ligand, one that acts as a HIV-1 entry inhibitor and a CXCR4 drug delivery targeting ligand. [ABSTRACT FROM AUTHOR]

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal—to design an entry inhibitor with improved drug-like qualities—and warrants expanded studies to achieve this.

Graphical abstract Highlights • Our study provides a new insight into the functional role of arachidonic acid during the formation of HIV-1 gp41 six-helix bundle. • Molecular docking techniques and isotherm titration microcalorimetry are adopted to explore the mechanism of kinetics. • AA presents the inhibitory activity to HIV-1JR-FL. Abstract The gp41 transmembrane domain of the envelope glycoprotein of the human immunodeficiency virus (HIV) modulates the conformation of the viral envelope spike. During the HIV fusion process, C-terminal heptad repeat (CHR, C34) wrap antiparallel to the N-terminal heptad repeat (NHR, N36) helices to form a stable six-helix bundle (6-HB) core structure, which brings the viral and cell membranes into close proximity for fusion. Therefore, inhibiting the formation of 6-HB is considered to be a key activity of an effective HIV-1 fusion inhibitor. The level of arachidonic acid (AA) is increased in HIV infected patients. Our study provides a new insight into the functional role of AA during the formation of HIV-1 gp41 6-HB. Native polyacrylamide gel electrophoresis (N-PAGE), enzyme-linked-immunosorbent serologic assay (ELISA) and circular dichroism (CD) spectroscopy were used to investigate the inhibition of AA for the formation of 6-HB. Molecular docking technique was adopted to explore the underlying mechanism. HIV-1 JR-FL (R5 strain) Envelope was adopted to determine the inhibition effect of AA. AA is shown to interfere with the formation of α-helical complexes of N36 and C34 by N-PAGE, ELISA and CD spectroscopy. The isotherm titration microcalorimetry (ITC) results indicate there is a single class of binding site on N36. ΔH and ΔS are −12.43 kJ mol−1 and 70.07 J mol−1 K−1, respectively, indicating hydrophobic interaction and electrostatic forces are the main acting forces. The molecular docking results manifest that AA interacts with the hydrophobic residues (Trp-571, Leu-568, Val-570 and Leu-576) and ionic interactions occur between Arg-579 and the -COOH of AA. The inhibitory activity of AA on HIV-1 JR-FL is quantified by 50% effective concentration (EC 50) and 90% effective concentration (EC 90), which are 31.42 ± 1.08 and 133.47 ± 18.10 μg mL−1, respectively. All the results indicate that AA is able to inhibit the formation of 6-HB but cannot disrupt the preformed 6-HB. Therefore, AA is a potential inhibitor for the viral fusion/entry. [ABSTRACT FROM AUTHOR]

Objectives We recently demonstrated that both 3-hydroxyphthalic anhydride (HP)- and maleic anhydride-modified chicken ovalbumin (OVA) could effectively inhibit HIV-1 infection. But because OVA may cause allergy in some human subjects, here we replaced OVA with human serum albumin (HSA) in designing a new anti-HIV-1 agent, HP-HSA, and then tested its anti-HIV-1 activity and cytotoxicity. Methods The in vitro anti-HIV-1 activities of HP-HSA were detected by measuring p24 production and luciferase activity. The cytotoxicities of HP-HSA on target cells and human vaginal and cervical epithelial cells and the effect of HP-HSA on human peripheral blood mononuclear cell (PBMC) proliferation were evaluated by XTT assay. The effect of HP-HSA on interferon-γ secretion by PBMCs was detected by enzyme-linked immunospot (ELISPOT) assay. Results We found that HP-HSA exhibited broad and potent antiviral activity against infection by the HIV-1 strains tested, including drug-resistant strains. HP-HSA displayed no or low cytotoxicity on human vaginal and cervical epithelial cells and the cells used for testing HIV-1 infectivity. In addition, HP-HSA had no significant effect on proliferation or interferon-γ secretion by normal or phytohaemagglutinin-stimulated human PBMCs. A time-of-addition assay indicated that HP-HSA was an HIV-1 entry inhibitor. Conclusions Because of its broad and potent anti-HIV-1 activity, low cytotoxicity and low immunogenicity to humans, HP-HSA has great potential for further development as a microbicide to prevent the sexual transmission of HIV. [ABSTRACT FROM PUBLISHER]

HIV-1 fusion with its target cells is mediated by the glycoprotein 41 (gp41) transmembrane subunit of the viral envelope glycoprotein (ENV). The current models propose that gp41 undergoes several conformational changes between the apposing viral and cell membranes to facilitate fusion. In this review we focus on the progress that has been made in revealing the dynamic role of the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions within gp41 to the fusion process. The involvement of these regions in the formation of the gp41 pre-hairpin and hairpin conformations during an ongoing fusion event was mainly discovered by their derived inhibitory peptides. For example, the core structure within the hairpin conformation in a dynamic fusion event is suggested to be larger than its high resolution structure and its minimal boundaries were determined in situ. Also, inhibitory peptides helped reveal the dual contribution of the NHR to the fusion process. Finally, we will also discuss several developments in peptide design that has led to a deeper understanding of the mechanism of viral membrane fusion. [ABSTRACT FROM AUTHOR]

The interactions between the N- and C-terminal heptad repeat (NHR and CHR) regions of the human immunodeficiency virus (HIV-1) glycoprotein gp41 create a structure comprising a 6-helix bundle (SHB). A sequence in the SHB named die "pocket" is crucial for die SHB's stability and for die fusion inhibitory activity of 36-residue NHR peptide N36. We report that a short 27-residue peptide, N27, which lacks die pocket sequence, exhibits potent inhibitory activity in both cell-cell and virus-cell fusion assays when fatty acids were conjugated to its N but not C terminus. Furthermore, mutations in the positions that prevent interaction with die CHR but not with the NHR resulted in a dramatic reduction in N27 activity. These data support a mechanism in which N27 mainly targets die CHR rather than die internal NHR coiled-coil, reveal the N-terminal edge of die endogenous core structure in situ and hence complement our recent findings of die C-terminal edge of die core, and provide a new approach for designing short inhibitors from die NHR region of other lentiviruses due to similarities in their envelope proteins. [ABSTRACT FROM AUTHOR]

Abstract: By using functionality inversion approach, we identified a new scaffold containing (S)-α-phenyl-γ-amino butanamide as CCR5 antagonists derived from the 1,3-propanediamine carboxamide pharmacophore protocol. The (2S)-2-phenyl-4-(8-aza-bicyclo[3.2.1]octan-8-yl)-butanamide derivatives display significantly high potency to antagonize CCR5 receptor with nanomolar IC50 values. [Copyright &y& Elsevier]

Abstract: The effects of introducing simple halogen, alkyl, and alkoxy substituents to the 4, 5, 6 and 7 positions of 1-(4-benzoylpiperazin-1-yl)-2-(1H-indol-3-yl)ethane-1,2-dione, an inhibitor of the interaction between HIV gp120 and host cell CD4 receptors, on activity in an HIV entry assay was examined. Small substituents at C-4 generally resulted in increased potency whilst substitution at C-7 was readily tolerated and uniformly produced more potent HIV entry inhibitors. Substituents deployed at C-6 and, particularly, C-5 generally produced a modest to marked weakening of potency compared to the prototype. Small alkyl substituents at N-1 exerted minimal effect on activity whilst increasing the size of the alkyl moiety led to progressively reduced inhibitory properties. These studies establish a basic understanding of the indole element of the HIV attachment inhibitor pharmacophore. [Copyright &y& Elsevier]

Abstract: We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose-dependent inhibition on HIV-1 fusion core formation with EC50s of 66–58nM, with no detectable toxicity. Our findings on effects of HIV-1 integrase are reported in the subsequent article. [Copyright &y& Elsevier]

4-Substituted-4-aminopiperidine is an interesting structural motif found in a number of bioactive compounds. An efficient and convenient method for the synthesis of 4-differently substituted-4-aminopiperidine derivatives was described, employing isonipecotate as a starting material and Curtius rearrangement as a key step. The alkylation of isonipecotate could introduce various substituents at the 4-position of the piperidine ring. With this key building block, we are able to efficiently synthesize piperazino-piperidine based CCR5 antagonist in a highly convergent manner free of using toxic reagents such as diethylaluminum cyanide. The concise synthesis of a potent bioavailable CCR5 antagonist as HIV-1 entry inhibitor, Sch-350634 (1) was accomplished in excellent yield using N′-Boc-4-methyl-4-aminopiperidine 5a as a smart building block. The new methodology provides a facile and practical access to the piperazino-piperidine amide analogs as HIV-1 entry inhibitors. [Copyright &y& Elsevier]

Fostemsavir/temsavir is an investigational HIV-1 entry inhibitor currently in late-stage clinical trials. Although it holds promise to be a first-in-class Env-targeted entry inhibitor for the clinic, issues with bioavailability relegate its use to salvage therapies only. As such, the development of a small molecule HIV-1 entry inhibitor that can be used in standard combination antiretroviral therapy (cART) remains a longstanding goal for the field. We previously demonstrated the ability of extending the chemotypes available to this class of inhibitor as the first step towards this overarching goal. In addition to poor solubility, metabolic stability is a crucial determinant of bioavailability. Therefore, in this short communication, we assess the metabolic stabilities of five of our novel chemotype entry inhibitors. We found that changing the piperazine core region of temsavir alters the stability of the compound in human liver microsome assays. Moreover, we identified an entry inhibitor with more than twice the metabolic stability of temsavir and demonstrated that the orientation of the core replacement is critical for this increase. This work further demonstrates the feasibility of our long-term goal—to design an entry inhibitor with improved drug-like qualities—and warrants expanded studies to achieve this. [ABSTRACT FROM AUTHOR]

We report here the development and optimization of a process synthesis for the HIV-1 entry inhibitor BNM-III-170 bis-TFA salt ( 1 ). The synthesis features a dynamic-kinetic resolution (DKR) to establish the initial stereogenicity. By taking advantage of significant sequence modifications of our first generation synthesis, inconjunction with the low solubility of late-stage intermediates, the overall efficiency of the synthesis has been significantly improved, now to proceed in an overall yield of 9.64% for the 16-steps, requiring only a single chromatographic separation., Competing Interests: The authors declare to have no competing financial interests.