Penetrating the Blood-Brain Barrier with New Peptide-Porphyrin Conjugates Having anti-HIV Activity.

Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide-drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide-porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.

Structural determinants conferring unusual long life in human serum to rattlesnake-derived antimicrobial peptide Ctn[15-34].

Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.

Repurposing anti-cancer porphyrin derivative drugs to target SARS-CoV-2 envelope.

Antiviral medicines to treat COVID-19 are still scarce. Porphyrins and porphyrin derivatives (PDs) usually present broad-spectrum antiviral activity with low risk of resistance development. In fact, some PDs are clinically approved to be used in anti-cancer photodynamic therapy and repurposing clinically approved PDs might be an alternative to treat COVID-19. Here, we characterize the ability of temoporfin, verteporfin, talaporfin and redaporfin to inactivate SARS-CoV-2 infectious particles. PDs light-dependent and -independent effect on SARS-CoV-2 infectivity were evaluated. PDs photoactivation successfully inactivated SARS-CoV-2 with very low concentrations and light dose. However, only temoporfin and verteporfin inactivated SARS-CoV-2 in the dark, being verteporfin the most effective. PDs treatment reduced viral load in infected Caco-2 cells, while not inducing cytotoxicity. Furthermore, light-independent treatment with temoporfin and verteporfin act on early stages of viral infection. Using lipid vehicles as membrane models, we characterized PDs interaction to the viral envelope. Verteporfin presented the lowest IC50 for viral inactivation and the highest partition coefficients (Kp) towards lipid bilayers. Curiously, although temoporfin and redaporfin presented similar Kps, redaporfin did not present light-independent antiviral activity, and only temoporfin and verteporfin caused lipid membrane disorder. In fact, redaporfin is located closer to the bilayer surface, while temoporfin and verteporfin are located closer to the centre. Our results suggest that viral envelope affinity, with penetration and destabilization of the lipid bilayer, seems critical to mediate PDs antiviral activity. Altogether, these findings open new avenues for the off-label application of temoporfin and verteporfin in the systemic treatment of COVID-19.

Parainfluenza Fusion Peptide Promotes Membrane Fusion by Assembling into Oligomeric Porelike Structures.

Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition.

Targeting Zika Virus with New Brain- and Placenta-Crossing Peptide-Porphyrin Conjugates.

Viral disease outbreaks affect hundreds of millions of people worldwide and remain a serious threat to global health. The current SARS-CoV-2 pandemic and other recent geographically- confined viral outbreaks (severe acute respiratory syndrome (SARS), Ebola, dengue, zika and ever-recurring seasonal influenza), also with devastating tolls at sanitary and socio-economic levels, are sobering reminders in this respect. Among the respective pathogenic agents, Zika virus (ZIKV), transmitted by Aedes mosquito vectors and causing the eponymous fever, is particularly insidious in that infection during pregnancy results in complications such as foetal loss, preterm birth or irreversible brain abnormalities, including microcephaly. So far, there is no effective remedy for ZIKV infection, mainly due to the limited ability of antiviral drugs to cross blood-placental and/or blood-brain barriers (BPB and BBB, respectively). Despite its restricted permeability, the BBB is penetrable by a variety of molecules, mainly peptide-based, and named BBB peptide shuttles (BBBpS), able to ferry various payloads (e.g., drugs, antibodies, etc.) into the brain. Recently, we have described peptide-porphyrin conjugates (PPCs) as successful BBBpS-associated drug leads for HIV, an enveloped virus in which group ZIKV also belongs. Herein, we report on several brain-directed, low-toxicity PPCs capable of targeting ZIKV. One of the conjugates, PP-P1, crossing both BPB and BBB, has shown to be effective against ZIKV (IC50 1.08 µM) and has high serum stability (t1/2 ca. 22 h) without altering cell viability at all tested concentrations. Peptide-porphyrin conjugation stands out as a promising strategy to fill the ZIKV treatment gap.

Structure-Stability-Function Mechanistic Links in the Anti-Measles Virus Action of Tocopherol-Derivatized Peptide Nanoparticles.

Measles remains one of the leading causes of child mortality worldwide and is re-emerging in some countries due to poor vaccine coverage, concomitant with importation of measles virus (MV) from endemic areas. The lack of specific chemotherapy contributes to negative outcomes, especially in infants or immunodeficient individuals. Fusion inhibitor peptides derived from the MV Fusion protein C-terminal Heptad Repeat (HRC) targeting MV envelope fusion glycoproteins block infection at the stage of entry into host cells, thus preventing viral multiplication. To improve efficacy of such entry inhibitors, we have modified a HRC peptide inhibitor by introducing properties of self-assembly into nanoparticles (NP) and higher affinity for both viral and cell membranes. Modification of the peptide consisted of covalent grafting with tocopherol to increase amphipathicity and lipophilicity (HRC5). One additional peptide inhibitor consisting of a peptide dimer grafted to tocopherol was also used (HRC6). Spectroscopic, imaging, and simulation techniques were used to characterize the NP and explore the molecular basis for their antiviral efficacy. HRC5 forms micellar stable NP while HRC6 aggregates into amorphous, loose, unstable NP. Interpeptide cluster bridging governs NP assembly into dynamic metastable states. The results are consistent with the conclusion that the improved efficacy of HRC6 relative to HRC5 can be attributed to NP instability, which leads to more extensive partition to target membranes and binding to viral target proteins.