Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment.

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane.

Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism.

The delivery of therapeutic molecules to the central nervous system is hampered by poor delivery across the blood-brain barrier (BBB). Several strategies have been proposed to enhance transport into the brain, including invasive techniques and receptor-mediated transport (RMT). Both approaches have several drawbacks, such as BBB disruption, receptor saturation, and off-target effects, raising safety issues. Herein, we show that specific domains of Dengue virus type 2 capsid protein (DEN2C) can be used as trans-BBB peptide vectors. Their mechanism of translocation is receptor-independent and consistent with adsorptive-mediated transport (AMT). One peptide in particular, named PepH3, reaches equilibrium distribution concentrations across the BBB in less than 24 h in a cellular in vitro assay. Importantly, in vivo biodistribution data with radiolabeled peptide derivatives show high brain penetration. In addition, there is fast clearance from the brain and high levels of excretion, showing that PepH3 is a very good candidate to be used as a peptide shuttle taking cargo in and out of the brain.

Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide.

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide's free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies.

Endothelium-Mediated Action of Analogues of the Endogenous Neuropeptide Kyotorphin (Tyrosil-Arginine): Mechanistic Insights from Permeation and Effects on Microcirculation.

Kyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (l-Tyr-l-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of d amino acid residues. Intravital microscopy data show that KTP-NH2 having a d-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 µM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 µM increase leukocyte rolling up to nearly 5-fold after 30 min, suggesting a proinflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.

Amidated and Ibuprofen-Conjugated Kyotorphins Promote Neuronal Rescue and Memory Recovery in Cerebral Hypoperfusion Dementia Model.

Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer's disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.

Correlation between membrane translocation and analgesic efficacy in kyotorphin derivatives.

Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) causes analgesia when systemically administered. The lipophilic ibuprofen-conjugated derivative of KTP-NH2 has improved analgesic efficacy. However, fast degradation by peptidases impacts negatively in the pharmacodynamics of these drugs. In this work, selected derivatives of KTP and KTP-NH2 were synthesized to combine lipophilicity and resistance to enzymatic degradation. Eight novel structural modifications were tested for the potential to transverse lipid membranes and to evaluate their efficacy in vivo. The rationale behind the design of the pool of the eight selected molecules consisted in the addition of individual group at the N-terminus, namely the tert-butyloxycarbonyl (Boc), γ-aminobutyric acid (GABA), acetyl, butanoyl, and propanoyl or in the substitution of the tyrosine residue by an indole moiety and in the replacement of the peptidic bond by a urea-like bond in some cases. All the drugs used in the study are intrinsically fluorescent, which enables the use of spectrofluorimetry to sample the drugs in the permeation assays. The results show that the BOC and indolyl derivatives of KTP-NH2 have maximal ability to permeate membranes with concomitant maximal analgesic power. Overall, the results demonstrate that membrane permeation is correlated with analgesic efficacy. However, this is not the only factor accounting for analgesia. KTP-NH2 for instance has low passive permeation but is known to have central action. In this case, hypothetical transcytosis over the blood-brain barrier seems to depend on dipeptide transporters.

Chemical conjugation of the neuropeptide kyotorphin and ibuprofen enhances brain targeting and analgesia.

The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 µmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.

Efeitos da ação antrópica sobre a hidrogeoquímica do rio Negro na orla de Manaus/AM

The Negro river, on the shoreline of the city of Manaus, receives an input of industrial and domestic wastes from its urban tributaries, which varying composition has been changing the natural characteristics of its waters. Aiming to assess the effect of the human action on the Negro River water samples were collected, and pH, Eh, electric conductivity and alkalinity (by potentiometry); dissolved oxygen (titrimetry); ammonia and nitrite (spectrophotometry); cations Ca, Mg, Na and K, and the metals Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn (by atomic absorption spectroscopy, AAS) were analyzed. The results for pH, electric conductivity, dissolved oxygen, ammoniacal nitrogen and nitrite values, indicated a worsening water quality in the Negro river, at the mouths of the São Raimundo (FISR) and Educandos (FIE) streams, which are the two major urban tributaries and receptors of domestic wastes and industrial effluents and downstream from them (JIE). Metals predominate in the dissolved phase and, apart from copper and zinc, are mostly above the maximum desirable limit allowed by the Health Ministry for human consumption. This study showed that in spite of the human contribution from its urban tributaries, the Negro river, still maintains its ability for diluting pollutants, mainly during the period of high water.

O rio Negro, na orla de Manaus, recebe de seus tributários urbanos resíduos domésticos e industriais, cuja composição variada vem modificando as características naturais de suas águas. Com o intuito de avaliar o efeito da ação antrópica sobre o rio Negro foram coletadas amostras de água e analisadas as variáveis: pH, Eh, condutividade elétrica e alcalinidade (por potenciometria); oxigênio dissolvido (titrimetria); nitrogênio amoniacal e de nitritos (espectrofotometria); os cátions Ca, Mg, Na e K, e os metais Cd, Cu, Cr, Fe, Mn, Ni, Pb e Zn (por espectroscopia de absorção atômica - EAA). Os resultados obtidos para pH, condutividade, oxigênio dissolvido, nitrogênio amoniacal e nitritos mostram piora na qualidade da água do rio Negro, na foz dos igarapés São Raimundo (FISR) e Educandos (FIE) e a jusante destes (JIE), que são os dois maiores tributários urbanos e receptores de esgotos domésticos e efluentes industriais. Os metais predominaram na fase dissolvida e, a maior parte, está acima do limite máximo desejável, preconizado pelo ministério da saúde para águas destinadas ao abastecimento público, exceto cobre e zinco. O estudo mostrou que apesar da contribuição antrópica dos tributários urbanos, o rio Negro mantém, ainda, sua capacidade de diluir os poluentes, principalmente, no período de maior volume de água.