Suchergebnisse

Data de publicació electrònica: 28-02-2021 ; Proteolytic instability is a critical limitation for peptide-based products. Although significant efforts are devoted to stabilize sequences against proteases/peptidases in plasma/serum, such approaches tend to be rather empirical, unspecific, time-consuming, and frequently not cost-effective. A more rational and potentially rewarding alternative is to identify the chemical grounds of susceptibility to enzymatic degradation of peptides so that proteolytic resistance can be tuned by manipulation of key chemical properties. In this regard, we conducted a meta-analysis of literature published over the last decade reporting experimental data on the lifetimes of peptides exposed to proteolytic conditions. Our initial database contained 579 entries and was curated with regard to amino acid sequence, chemical modification, terminal half-life (t1/2 ) or other stability readouts, type of stability assay, and biological application of the study. Although the majority of entries in the database corresponded to (slightly or substantially) modified peptides, we chose to focus on unmodified ones, as we aimed to decipher intrinsic characteristics of peptide proteolytic susceptibility. Specifically, we developed a multivariable regression model to unravel those peptide properties with most impact on proteolytic stability and thus potential t1/2 predicting ability. Model validation was done by two different approaches. First, a library of peptides spanning a large interval of properties that modulate stability was synthesized and their t1/2 in human serum were experimentally determined. Second, the t1/2 of 21 selected peptides approved for clinical use or in clinical trials were recorded and matched with the model-estimated values. With both approaches, good correlation between experimental and predicted t1/2 data was observed. ; This research was supported by the Portuguese Fundação para a Ciência e a Tecnologia (FCT; grants PD/BD/128281/2017, PTDC/BBB‐NAN/1578/2014, PTDC/BIA‐VIR/29495/2017, UID/Multi/04349/2019, and PTDC/QUI‐NUC/30147/2017), the Spanish Ministry of Economy and Innovation (MINECO, grants AGL2014‐52395‐C2‐2‐R and AGL2017‐84097‐C2‐2‐R, and Maria de Maeztu Program for Centers of Excellence); the European Union H2020‐MSCA‐RISE‐2014 program (grant no. 828774), and the "La Caixa" Banking Foundation (grant HR17‐00409).

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood-brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides. ; The authors thank the Portuguese Funding Agency, Fundação para a Ciência e a Tecnologia, FCT IP, for financial support (grants: PD/BD/128281/2017, PTDC/BBB-NAN/1578/2014); European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 828774; and "la Caixa" Banking Foundation under the project code HR17-00409. JC and RS gratefully acknowledge the FCT for financial support through projects UID/Multi/04349/2019 and PTDC/QUI-NUC/30147/2017.

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. ; Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/52150011), by the European Union (H2020-FETOPEN-2018-2019-2020-01 grant no 828774), and by the Spanish Ministry of Science and Innovation (AEI/FEDER grant CTQ2017-84371-P). NMR experiments were performed in the "Manuel Rico" NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB. Additional funding from Fundação para a Ciência e Tecnologia (FCT-MCTES) is also acknowledged for DA Mendonça (PD/BD/136752/2018) and for C. Cruz-Oliveira and I. Cadima-Couto (PTDC/BIA-VIR/29495/2017).

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. ; Work supported by the La Caixa Health Foundation (project HR17_00409, ID 100010434, agreement LCF/PR/HR17/52150011) and by the European Union (H2020-FETOPEN-2018-2019-2020-01 grant no 828774). The Department of Medicine and Life Sciences, Pompeu Fabra University, belongs to the María de Maeztu network of Units of ...