Mechanism of multivalent nanoparticle encounter with HIV-1 for potency enhancement of peptide triazole virus inactivation.

Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal ß-amyloid fragment.

Soluble ß-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar ß-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within ß-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and ß-secretases, and resident carboxypeptidase. The N-terminal ß-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length ß-amyloid, the N-terminal ß-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal ß-amyloid fragment proved to be highly potent and more effective than full-length ß-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal ß-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length ß-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal ß-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal ß-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal ß-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

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.

Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions.

BACKGROUND: We examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity. RESULTS: KR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions. CONCLUSIONS: The antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion.

Chimeric Cyanovirin-MPER recombinantly engineered proteins cause cell-free virolysis of HIV-1.

Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.

A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor α.

Human interleukin-5 receptor α (IL5Rα) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5Rα resulted in complete loss of IL5 binding. To localize the functionally important carbohydrate moieties, we employed site-directed mutagenesis at the N-glycosylation sites (Asn(15), Asn(111), Asn(196), and Asn(224)). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability-preserving changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5Rα and other cytokine receptors. These approaches were most successful at Asn(15), Asn(111), and Asn(224). In contrast, any replacement at Asn(196) severely reduced stability, with the N196T mutant having a reduced binding affinity for IL5 and diminished biological activity because of the lack of cell surface expression. Lectin inhibition analysis suggested that the carbohydrate at Asn(196) is unlikely involved in direct ligand binding. Taking this into account, we constructed a stable variant, with triple mutational deglycosylation (N15D, I109V/V110T/N111D, and L223R/N224Q). The re-engineered protein retained Asn(196) while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5Rα, thus demonstrating a unique role for Asn(196) glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.

Conformational and structural features of HIV-1 gp120 underlying the dual receptor antagonism by cross-reactive neutralizing antibody m18.

We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.

Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120.

We evaluated the potential of a quartz crystal microbalance with dissipation monitoring (QCM-D) to provide a sensitive, label-free method for detecting the conformational rearrangement of glycoprotein gp120 upon binding to different ligands. This glycoprotein is normally found on the envelope of the HIV-1 virus and is involved in viral entry into host cells. It was immobilized on the surface of the sensing element of the QCM-D and was exposed to individual solutions of several different small-molecule inhibitors as well as to a solution of a soluble form of the host cell receptor to which gp120 binds. Instrument responses to ligand-triggered changes were in qualitative agreement with conformational changes as suggested by other biophysical methods.

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.

The active core in a triazole peptide dual-site antagonist of HIV-1 gp120.

In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co-receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy- and amino-terminal residues from the parent 12-residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)-Trp cluster at residues 6 and 7 with ferrocenyl-triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino-terminal residues distal to the central triazole(Pro)-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.

Modular, self-assembling peptide linkers for stable and regenerable carbon nanotube biosensor interfaces.

As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.