Palmitic Acid Analogs Exhibit Nanomolar Binding Affinity for the HIV-1 CD4 Receptor and Nanomolar Inhibition of gp120-to-CD4 Fusion

Background: We recently reported that palmitic acid (PA) is a novel and efficient CD4 fusion inhibitor to HIV-1 entry and infection. In the present report, based on in silico modeling of the novel CD4 pocket that binds PA, we describe discovery of highly potent PA analogs with increased CD4 receptor binding affinities (Kd) and gp120-to-CD4 inhibition constants (Ki). The PA analogs were selected to satisfy Lipinski’s rule of drug-likeness, increased solubility, and to avoid potential cytotoxicity. Principal Findings: PA analog 2-bromopalmitate (2-BP) was most efficacious with Kd,74 nM and Ki,122 nM, ascorbyl palmitate (6-AP) exhibited slightly higher Kd,140 nM and Ki,354 nM, and sucrose palmitate (SP) was least efficacious binding to CD4 with Kd,364 nM and inhibiting gp120-to-CD4 binding with Ki,1486 nM. Importantly, PA and its analogs specifically bound to the CD4 receptor with the one to one stoichiometry. Significance: Considering observed differences between K i and K d values indicates clear and rational direction for improving inhibition efficacy to HIV-1 entry and infection. Taken together this report introduces a novel class of natural small molecules fusion inhibitors with nanomolar efficacy of CD4 receptor binding and inhibition of HIV-1 entry

Inhibition of highly productive HIV-1 infection in T cells, primary human macrophages, microglia, and astrocytes by Sargassum fusiforme

BACKGROUND: The high rate of HIV-1 mutation and increasing resistance to currently available antiretroviral (ART) therapies highlight the need for new antiviral agents. Products derived from natural sources have been shown to inhibit HIV-1 replication during various stages of the virus life cycle, and therefore represent a potential source of novel therapeutic agents. To expand our arsenal of therapeutics against HIV-1 infection, we investigated aqueous extract from Sargassum fusiforme (S. fusiforme) for ability to inhibit HIV-1 infection in the periphery, in T cells and human macrophages, and for ability to inhibit in the central nervous system (CNS), in microglia and astrocytes. RESULTS: S. fusiforme extract blocked HIV-1 infection and replication by over 90% in T cells, human macrophages and microglia, and it also inhibited pseudotyped HIV-1 (VSV/NL4-3) infection in human astrocytes by over 70%. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5)-tropic HIV-1, was dose dependant and long lasting, did not inhibit cell growth or viability, was not toxic to cells, and was comparable to inhibition by the nucleoside analogue 2', 3'-didoxycytidine (ddC). S. fusiforme treatment blocked direct cell-to-cell infection spread. To investigate at which point of the virus life cycle this inhibition occurs, we infected T cells and CD4-negative primary human astrocytes with HIV-1 pseudotyped with envelope glycoprotein of vesicular stomatitis virus (VSV), which bypasses the HIV receptor requirements. Infection by pseudotyped HIV-1 (VSV/NL4-3) was also inhibited in a dose dependant manner, although up to 57% less, as compared to inhibition of native NL4-3, indicating post-entry interferences. CONCLUSION: This is the first report demonstrating S. fusiforme to be a potent inhibitor of highly productive HIV-1 infection and replication in T cells, in primary human macrophages, microglia, and astrocytes. Results with VSV/NL4-3 infection, suggest inhibition of both entry and post-entry events of the virus life cycle. Absence of cytotoxicity and high viability of treated cells also suggest that S. fusiforme is a potential source of novel naturally occurring antiretroviral compounds that inhibit HIV-1 infection and replication at more than one site of the virus life cycle

Inhibition of HIV-1 infection in ex vivo cervical tissue model of human vagina by palmitic acid; implications for a microbicide development.

Approximately 80% of all new HIV-1 infections are acquired through sexual contact. Currently, there is no clinically approved microbicide, indicating a clear and urgent therapeutic need. We recently reported that palmitic acid (PA) is a novel and specific inhibitor of HIV-1 fusion and entry. Mechanistically, PA inhibits HIV-1 infection by binding to a novel pocket on the CD4 receptor and blocks efficient gp120-to-CD4 attachment. Here, we wanted to assess the ability of PA to inhibit HIV-1 infection in cervical tissue ex vivo model of human vagina, and determine its effect on Lactobacillus (L) species of probiotic vaginal flora.Our results show that treatment with 100-200 µM PA inhibited HIV-1 infection in cervical tissue by up to 50%, and this treatment was not toxic to the tissue or to L. crispatus and jensenii species of vaginal flora. In vitro, in a cell free system that is independent of in vivo cell associated CD4 receptor; we determined inhibition constant (Ki) to be ∼2.53 µM.These results demonstrate utility of PA as a model molecule for further preclinical development of a safe and potent HIV-1 entry microbicide inhibitor

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against \u3ci\u3eBacillus\u3c/i\u3e Species

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-L-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature

Nanotechnologies for biosensor and biochip (Editorial)

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PA inhibition constant for gp120-to-CD4 complex formation.

<p>Inhibition of gp120-CD4 complex formation was investigated by gp120 capture ELISA. Envelope gp120 (IIIB) protein was captured on 96 well flat bottom plates, washed, and incubated in the presence of CD4-biotin alone or in the presence of increasing concentrations of PA, as indicated. Strepavidin-HRP was added, and then developed by addition of o-Phenylenediamine dihydrochloride (OPD) substrate. Colorimetric reaction was stopped by adding 1 N HCl, and read at 490 nm. % CD4 binding was calculated from gp120-CD4 complex formation in the absence of PA inhibitor, and inhibition constant, K_i, was calculated utilizing previously published equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024803#pone.0024803-Cheng1" target="_blank">[18]</a>. Representative of three experiments, all data are mean ± SD.</p

Inhibition of HIV-1 infection in human cervix model of vaginal mucosa.

<p>3 mm³ biopsy punches of the ectocervix tissue samples from premenopausal women with conditions not involving the cervix were processed within 1–3 hours after surgery and directly cultured in 48 well plates in 300 µl/well DMEM/F12 media. (A) Paraffin embedded, and hematoxylin and eosin (H&E) stained sections of the uninfected ectocervix tissue were identified to be composed of (a) stratified squamous epithelial cell layer, (b) basal epithelial layer, and (c) submocosa, which was visualized on an Olympus BX41 Altra 20 Soft Image System, 100× magnification. (B) Replicates (n = 6) of tissue were treated for 24 h with 0, 100, or 200 µM PA, and then infected with 2×10⁵ p24/ml cell-free HIV-1 BaL in 300 µl for 16 h. Tissues was washed 3 times to remove the virus, and returned to culture with each respective treatment for the duration of the experiment. At the indicated time points, HIV-1 replication was tested by p24 ELISA, and repeated measures ANOVA was used to calculate statistical significance (*) between groups. (C) At day 10 after infection, tissue was collected and viability determined by the MTT assay. Representative of three experiments, all data are mean ± SD.</p

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

<it>Sargassum fusiforme </it>fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

<p>Abstract</p> <p><it>Sargassum fusiforme </it>(Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on <it>Sargassum fusiforme </it>mixture. Here, we report isolation of a bioactive fraction SP4-2 (<it>S. fusiforme</it>), which at 8 μg/ml inhibited HIV-1 infection by 86.9%, with IC₅₀value of 3.7 μg. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 μg/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that <it>S. fusiforme </it>inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that <it>S. fusiforme </it>is a lead candidate for anti-HIV-1 drug development.</p