HIV-1 Env gp120 structural determinants for peptide triazole dual receptor site antagonism.

Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide's affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development.

Fabrication of intelligent/active films based on chitosan/polyvinyl alcohol matrices containing Jacaranda cuspidifolia anthocyanin for real-time monitoring of fish freshness.

The present work describes the natural anthocyanin from Jacaranda cuspidifolia (JC) flower immobilized within a biopolymer matrix composed of chitosan (CS) and polyvinyl alcohol (PVA) gave novel intelligent/active packaging films (CPC). We introduced microwave irradiation to prepare polymeric composite films noticed faster mixing of the polymers and extract take place than the conventional method. The prepared composite films are characterized by various analytical and spectroscopic techniques. The smooth SEM images demonstrated CS/PVA matrix miscibility and compatibility with anthocyanin for the film formation. The addition of anthocyanin to the CS/PVA films significantly reduced UV-Vis light transmission, while causing a slight decrease in the films transparency. An increased anthocyanin concentration on polymer films showed improved oxygen permeability (77.09 %), moisture retention capacity (11.64 %), and water vapor transmission rate (43.10 %) substantially. Additionally, the prepared CPC smart films exhibited strong antioxidant (97.92 %) as well as antibacterial activities against common foodborne pathogens such as S. aureus, and E. coli. Furthermore, the prepared smart films demonstrated pink color in acidic, while grey to yellowish in basic solvent. Further, the color response of the freshness label was consistent with the spoilage Total Volatile Basic-Nitrogen (TVB-N) content determined in the fish samples with varied time period. The CPC smart films also showed promising application in terms of monitoring freshness of the fish fillets at room temperature. The obtained results suggested that, the prepared CPC smart films have potential to be used as quality indicator in the marine food packaging system.

Green synthesis of 3-methyl-4-(hetero)aryl methylene isoxazole-5(4H)-ones using WEOFPA/glycerol: evaluation of anticancer and electrochemical behaviour properties.

In the present study, the MCRs of hydroxylamine hydrochloride and ethyl acetoacetate with various substituted aromatic and heteroaromatic aldehydes reacted to produce 3-methyl-4-(hetero)aryl methylene isoxazole-5(4H)-ones derivatives catalyzed by an agro-waste-based solvent medium in an oil bath at 60 °C with stirring. The developed protocol features several advantages, such as being benign and eco-friendly, efficient, avoiding the use of hazardous solvents, and inexpensive, while giving isoxazole derivatives in an 86-92% yield. The homogeneity of the product was confirmed by various spectroscopic analyses. Further, in vitro anticancer evaluation of the synthesized compounds (4h-4o) against lung cancer cells was performed, and among them, 4j, 4k, 4m, and 4o exhibited excellent anticancer activity and compounds 4i and 4n moderate inhibitory activity against lung cancer A549 cells to the reference drug doxorubicin. Furthermore, the derivatives were subjected to electrochemical behaviour studies using cyclic voltammetry and they showed intensive oxidation and reduction potential and also showed excellent anti-oxidant agents. Based on this research study, more and more novel structures of isoxazoles are being designed and synthesized, and their electrochemical behaviour and anticancer activities are studied for the development of novel drug-like candidates.

Targeting cell surface HIV-1 Env protein to suppress infectious virus formation.

HIV-1 Env protein is essential for host cell entry, and targeting Env remains an important antiretroviral strategy. We previously found that a peptide triazole thiol KR13 and its gold nanoparticle conjugate AuNP-KR13 directly and irreversibly inactivate the virus by targeting the Env protein, leading to virus gp120 shedding, membrane disruption and p24 capsid protein release. Here, we examined the consequences of targeting cell-surface Env with the virus inactivators. We found that both agents led to formation of non-infectious virus from transiently transfected HEK293T cells. The budded non-infectious viruses lacked Env gp120 but contained gp41. Importantly, budded virions also retained the capsid protein p24, in stark contrast to p24 leakage from viruses directly treated by these agents and arguing that the agents led to deformed viruses by transforming the cells at a stage before virus budding. We found that the Env inactivators caused gp120 shedding from the transiently transfected HEK293T cells as well as non-producer CHO-K1-gp160 cells. Additionally, AuNP-KR13 was cytotoxic against the virus-producing HEK293T and CHO-K1-gp160 cells, but not untransfected HEK293T or unmodified CHO-K1 cells. The results obtained reinforce the argument that cell-surface HIV-1 Env is metastable, as on virus particles, and provides a conformationally vulnerable target for virus suppression and infectious cell inactivation.

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection.

Non-natural peptide triazole antagonists of HIV-1 envelope gp120.

We investigated the derivation of non-natural peptide triazole dual receptor site antagonists of HIV-1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG-156 [R-I-N-N-I-X-W-S-E-A-M-M-CONH(2), in which X=ferrocenyltriazole-Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co-receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG-156, typified by UM-24 (Cit-N-N-I-X-W-S-CONH(2)) and containing the critical central stereospecific (L)X-(L)W cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM-24 to the greatest extent possible. The analogue with the critical "hot spot" residue Trp 6 replaced with L-3-benzothienylalanine (Bta) (KR-41), as well as a completely non-natural analogue containing D-amino acid substitutions outside the central cluster (KR-42, (D)Cit-(D)N-(D)N-(D)I-X-Bta-(D)S-CONH(2)), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV-1 Env gp120.