RESUMEN
The search for superconductivity in infinite-layer nickelates was motivated by analogy to the cuprates, and this perspective has framed much of the initial consideration of this material. However, a growing number of studies have highlighted the involvement of rare-earth orbitals; in that context, the consequences of varying the rare-earth element in the superconducting nickelates have been much debated. Here, we show notable differences in the magnitude and anisotropy of the superconducting upper critical field across the La-, Pr-, and Nd-nickelates. These distinctions originate from the 4f electron characteristics of the rare-earth ions in the lattice: They are absent for La3+, nonmagnetic for the Pr3+ singlet ground state, and magnetic for the Nd3+ Kramer's doublet. The unique polar and azimuthal angle-dependent magnetoresistance found in the Nd-nickelates can be understood to arise from the magnetic contribution of the Nd3+ 4f moments. Such robust and tunable superconductivity suggests potential in future high-field applications.
RESUMEN
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.
Asunto(s)
Antivirales/farmacología , VIH-1/fisiología , Péptidos/farmacología , Triazoles/farmacología , Ensamble de Virus/efectos de los fármacos , Inactivación de Virus , Productos del Gen env del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Animales , Células CHO , Cricetulus , Células HEK293 , HumanosRESUMEN
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.
Asunto(s)
Disulfuros/farmacología , Proteína gp120 de Envoltorio del VIH/química , VIH-1/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , Inactivación de Virus , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Dimerización , Disulfuros/química , Relación Dosis-Respuesta a Droga , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Simulación del Acoplamiento Molecular , Mutación , Péptidos/química , Péptidos/farmacología , Compuestos de Sulfhidrilo/química , Triazoles/química , Triazoles/farmacologíaRESUMEN
We derived macrocyclic HIV-1 antagonists as a new class of peptidomimetic drug leads. Cyclic peptide triazoles (cPTs) retained the gp120 inhibitory and virus-inactivating signature of parent PTs, arguing that cyclization locked an active conformation. The six-residue cPT 9 (AAR029b) exhibited submicromolar antiviral potencies in inhibiting cell infection and triggering gp120 shedding that causes irreversible virion inactivation. Importantly, cPTs were stable to trypsin and chymotrypsin compared to substantial susceptibility of corresponding linear PTs.
Asunto(s)
Fármacos Anti-VIH/química , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Oligopéptidos/química , Péptidos Cíclicos/química , Triazoles/química , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Línea Celular , Quimotripsina/química , VIH-1/fisiología , Humanos , Modelos Moleculares , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Conformación Proteica , Triazoles/síntesis química , Triazoles/farmacología , Tripsina/química , Virión/efectos de los fármacos , Virión/fisiología , Internalización del VirusRESUMEN
We used coordinated mutagenesis, synthetic design, and flexible docking to investigate the structural mechanism of Env gp120 encounter by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated that the PT class of inhibitors suppresses binding at both CD4 and coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent irreversible virus inactivation. Despite these enticing anti-HIV-1 phenotypes, structural understanding of the PT-gp120 binding mechanism has been incomplete. Here we found that PT engages two inhibitor ring moieties at the junction between the inner and outer domains of the gp120 protein. The results demonstrate how combined occupancy of two gp120 cavities can coordinately suppress both receptor and coreceptor binding and conformationally entrap the protein in a destabilized state. The two-cavity model has common features with small molecule gp120 inhibitor binding sites and provides a guide for further design of peptidomimetic HIV-1 inactivators based on the PT pharmacophore.
Asunto(s)
Fármacos Anti-VIH/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/química , Péptidos/química , Peptidomiméticos/química , Triazoles/química , Fármacos Anti-VIH/farmacología , Anticuerpos Monoclonales/química , Sitios de Unión , Antígenos CD4/química , VIH-1/efectos de los fármacos , VIH-1/genética , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Mutación , Péptidos/farmacología , Peptidomiméticos/farmacología , Unión Proteica , Relación Estructura-Actividad , Termodinámica , Triazoles/farmacología , Inactivación de VirusRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Nanoconjugados/toxicidad , Péptidos/farmacología , Triazoles/farmacología , Fármacos Anti-VIH/síntesis química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Oro/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/crecimiento & desarrollo , Humanos , Nanoconjugados/ultraestructura , Tamaño de la Partícula , Péptidos/síntesis química , Unión Proteica , Triazoles/síntesis química , Inactivación de Virus/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host-cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT-gp120 complex as a blueprint to design a covalently conjugated PT-gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21-E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT-gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT-gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT-gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT-gp120 conjugate to the noncovalent PT-gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT-gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced "off" state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , VIH-1 , Péptidos/química , Triazoles/química , Anticuerpos Monoclonales/química , Sitios de Unión , Biotinilación , Antígenos CD4/química , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Proteína gp120 de Envoltorio del VIH/genética , Simulación de Dinámica Molecular , Mutación , Polietilenglicoles/químicaRESUMEN
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.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , Péptidos/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Triazoles/metabolismo , Virión/efectos de los fármacos , Inactivación de Virus , Antivirales/metabolismo , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Conformación ProteicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/ultraestructura , Microscopía Electrónica de Transmisión , Plásmidos , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genéticaRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Secuencia de Aminoácidos , Calorimetría , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/virología , VIH-1/metabolismo , Humanos , Peptidomiméticos/química , Peptidomiméticos/farmacología , Unión Proteica , Triazoles/química , Triazoles/farmacologíaAsunto(s)
Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Péptidos/química , Triazoles/química , Triazoles/farmacología , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular Tumoral , Oro/química , Proteína p24 del Núcleo del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Nanopartículas del Metal/químicaRESUMEN
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.