Towards the development of innovative highly effective dual action anti-HIV peptides

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Mechanisms of bacterial membrane permeabilization by crotalicidin Ctn and its fragment Ctn 15–34, antimicrobial peptides from rattlesnake venom

Publication . Peinado, Clara Pérez; Dias, Susana; Domingues, Marco André Manso; Benfield, Aurélie H.; Freire, João Miguel; Rádis-Baptista, Gandhi; Gaspar, Diana; Castanho, Miguel A. R. B.; Craik, David J.; Henriques, Sónia Troeira; Veiga, Ana Salome; Andreu, David

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

2018Journal article

Open access

Anti-HIV-1 activity of pepRF1, a proteolysis-resistant CXCR4 antagonist derived from Dengue virus capsid protein

Publication . Cadima Couto, Carla Iris; Tauzin, Alexandra; Freire, João Miguel; Figueira, Tiago N.; Silva, Rúben D. M.; Pérez-Peinado, Clara; Cunha-Santos, Catarina; Bártolo, Inês; Taveira, Nuno; Gano, Lurdes; Correia, João D. G.; Goncalves, Joao; Mammano, Fabrizio; Andreu, David; Castanho, Miguel A. R. B.; Veiga, Ana Salomé

There is an urgent need for the development of new anti-HIV drugs that can complement existing medicines to be used against resistant strains. Here, we report the anti-HIV-1 peptide pepRF1, a human serum-resistant peptide derived from the Dengue virus capsid protein. In vitro, pepRF1 shows a 50% inhibitory concentration of 1.5 nM with a potential therapeutic window higher than 53 000. This peptide is specific for CXCR4-tropic strains, preventing viral entry into target cells by binding to the viral coreceptor CXCR4, acting as an antagonist of this receptor. pepRF1 is more effective than T20, the only peptide-based HIV-1 entry inhibitor approved, and excels in inhibiting a HIV-1 strain resistant to T20. Potentially, pepRF1 can be used alone or in combination with other anti-HIV drugs. Furthermore, one can also envisage its use as a novel therapeutic strategy for other CXCR4-related diseases.

2020Journal article

Restricted access

Structure-stability-function mechanistic links in the anti-measles virus action of tocopherol-derivatized peptide nanoparticles

Publication . Figueira, Tiago Nascimento; Mendonça, Diogo A.; Gaspar, Diana; Melo, Manuel N.; Moscona, Anne; Porotto, Matteo; Castanho, Miguel A. R. B.; Veiga, Ana Salomé

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.

2018Journal article

Restricted access

Effective in vivo targeting of influenza virus through a cell-penetrating/fusion inhibitor tandem peptide anchored to the plasma membrane

Publication . Figueira, Tiago Nascimento; Augusto, Marcelo Tavares; Rybkina, K.; Stelitano, D.; Noval, M. G.; Harder, O. E.; Veiga, Ana Salome; Huey, D.; Alabi, C. A.; Biswas, S.; Niewiesk, S.; Moscona, A.; Santos, Nuno C.; Castanho, Miguel A. R. B.; Porotto, M.

The impact of influenza virus infection is felt each year on a global scale when approximately 5−10% of adults and 20−30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15−30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

2018Journal article

Restricted access

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms

Publication . Pinto, Sandra; Dias, Susana; Cruz, Ana F; Mil-Homens, Dalila; Fernandes, Fabio; Valle, Javier; Andreu, David; Prieto, Manuel; Castanho, Miguel A. R. B.; Coutinho, Ana; Veiga, Ana Salomé

Objectives: To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. Methods: Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. Results: Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. Conclusions: Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

2019Journal article

Open access