The mechanism of action of antimicrobial peptides : lipid vesicles vs. bacteria

Copyright © 2012 Melo and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.The authors acknowledge the European FP7-PEOPLE-2009-IEF-254559 grant to Manuel N. Melo and the Fundação para a Ciência e a Tecnologia (Portugal) project PTDC/QUI-BIQ/112929/2009

Pharmaceutical innovations: the grand challenges ahead

Copyright © 2020 Aroeira and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Lifestyles are evolving rapidly due to swiftly evolving technologies rapid exchange of information across the globe, and facilitated mobility of people across long distances. In addition, climate changes accelerate the geographical dynamics of disease. The result is that both communicable and non-communicable diseases pose challenges never faced before and the perception of the way pharmaceutical sciences are dealing with such changes is under unprecedented scrutiny. The discredit in science-based solutions has a tremendous societal impact and is detrimental to evidence-based pharmacology at large. Pharmaceutical innovation that target the needs of healthy living and meet the expectation of society are urgently needed and are a worthy effect of both industrial and academic researchers. A reflection on the grand challenges ahead in thus timely and appropriate.This work was supported by La Caixa Foundation (grant reference: IMM/BPD/107-2018 to RA).info:eu-repo/semantics/publishedVersio

Anticancer peptides : prospective innovation in cancer therapy

© Springer International Publishing Switzerland 2016Current cancer treatments require improvements in selectivity and efficacy. Surgery, radiation, and chemotherapy approaches result in patient’s suffering over time due to the development of severe side-effects that simultaneously condition adherence to therapy. Biologically active peptides, in particular antimicrobial peptides (AMPs), are versatile molecules in terms of biological activities. The cytotoxic activities of several AMPs turn this group of molecules into an amazing pool of new templates for anticancer drug development. However, several unmet challenges limit application of peptides in cancer therapy. The mechanism(s) of action of the peptides need better description and understanding, and innovative targets have to be discovered and explored, facilitating drug design and development. In this chapter, we explore the natural occurring AMPs as potential new anticancer peptides (ACPs) for cancer prevention and treatment. Their modes of action, selectivity to tumor compared to normal cells, preferential targets, and applications, but also their weaknesses, are described and discussed.info:eu-repo/semantics/publishedVersio

From antimicrobial to anticancer peptides : a review

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms. Although AMPs have been essentially studied and developed as potential alternatives for fighting infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been regarded as a therapeutic strategy to explore. As human cancer remains a cause of high morbidity and mortality worldwide, an urgent need of new, selective, and more efficient drugs is evident. Even though ACPs are expected to be selective toward tumor cells without impairing the normal body physiological functions, the development of a selective ACP has been a challenge. It is not yet possible to predict antitumor activity based on ACPs structures. ACPs are unique molecules when compared to the actual chemotherapeutic arsenal available for cancer treatment and display a variety of modes of action which in some types of cancer seem to co-exist. Regardless the debate surrounding the definition of structure-activity relationships for ACPs, great effort has been invested in ACP design and the challenge of improving effective killing of tumor cells remains. As detailed studies on ACPs mechanisms of action are crucial for optimizing drug development, in this review we provide an overview of the literature concerning peptides' structure, modes of action, selectivity, and efficacy and also summarize some of the many ACPs studied and/or developed for targeting different solid and hematologic malignancies with special emphasis on the first group. Strategies described for drug development and for increasing peptide selectivity toward specific cells while reducing toxicity are also discussed.The authors thank Fundação para a Ciência e a Tecnologia (FCT- MEC, Portugal) for funding—PTDC/QUI-BIO/112929/2009. Diana Gaspar also acknowledges FCT for fellowship SFRH/BPD/ 73500/2010 and A. Salome Veiga for funding within the FCT Investigator Programme (IF/00803/2012

Resazurin reduction-based assays revisited: guidelines for accurate reporting of relative differences on metabolic status

© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Cell viability and metabolic activity are ubiquitous parameters used in biochemistry, molecular biology, and biotechnological studies. Virtually all toxicology and pharmacological projects include at some point the evaluation of cell viability and/or metabolic activity. Among the methods used to address cell metabolic activity, resazurin reduction is probably the most common. At variance with resazurin, resorufin is intrinsically fluorescent, which simplifies its detection. Resazurin conversion to resorufin in the presence of cells is used as a reporter of metabolic activity of cells and can be detected by a simple fluorometric assay. UV–Vis absorbance is an alternative technique but is not as sensitive. In contrast to its wide empirical “black box” use, the chemical and cell biology fundamentals of the resazurin assay are underexplored. Resorufin is further converted to other species, which jeopardizes the linearity of the assays, and the interference of extracellular processes has to be accounted for when quantitative bioassays are aimed at. In this work, we revisit the fundamentals of metabolic activity assays based on the reduction of resazurin. Deviation to linearity both in calibration and kinetics, as well as the existence of competing reactions for resazurin and resorufin and their impact on the outcome of the assay, are addressed. In brief, fluorometric ratio assays using low resazurin concentrations obtained from data collected at short time intervals are proposed to ensure reliable conclusions.The project leading to these results has received funding from “la Caixa” Foundation and FCT, I.P. under the project code [LCF/PR/HR21/00605], BREAST-BRAIN-N-BBB.info:eu-repo/semantics/publishedVersio

The use of antibody-antibiotic conjugates to fight bacterial infections

Copyright © 2022 Cavaco, Castanho and Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The emergence of antimicrobial resistance (AMR) is rapidly increasing and it is one of the significant twenty-first century’s healthcare challenges. Unfortunately, the development of effective antimicrobial agents is a much slower and complex process compared to the spread of AMR. Consequently, the current options in the treatment of AMR are limited. One of the main alternatives to conventional antibiotics is the use of antibodyantibiotic conjugates (AACs). These innovative bioengineered agents take advantage of the selectivity, favorable pharmacokinetic (PK), and safety of antibodies, allowing the administration of more potent antibiotics with less off-target effects. Although AACs’ development is challenging due to the complexity of the three components, namely, the antibody, the antibiotic, and the linker, some successful examples are currently under clinical studies.The project leading to these results has received funding from “la Caixa” Foundation (ID 100010434), under the agreement LCF/PR/HR17/52150011 and from Portuguese Funding Agency, Fundação para a Ciência e Tecnologia (FCT IP, grants PD/BD/128281/2017 and DL 7/2016/CP1451/CT0023).info:eu-repo/semantics/publishedVersio

Pharmacological potential of the endogenous dipeptide kyotorphin and selected derivatives

Copyright © 2017 Perazzo, Castanho and Sá Santos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The endogenous peptide kyotorphin (KTP) has been extensively studied since it was discovered in 1979. The dipeptide is distributed unevenly over the brain but the majority is concentrated in the cerebral cortex. The putative KTP receptor has not been identified yet. As many other neuropeptides, KTP clearance is mediated by extracellular peptidases and peptide transporters. From the wide spectrum of biological activity of KTP, analgesia was by far the most studied. The mechanism of action is still unclear, but researchers agree that KTP induces Met-enkephalins release. More recently, KTP was proposed as biomarker of Alzheimer disease. Despite all that, KTP limited pharmacological value prompted researchers to develop derivatives more lipophilic and therefore more prone to cross the blood-brain barrier (BBB), and also more resistant to enzymatic degradation. Conjugation of KTP with functional molecules, such as ibuprofen, generated a new class of compounds with additional biological properties. Moreover, the safety profile of these derivatives compared to opioids and their efficacy as neuroprotective agents greatly increases their pharmacological value.Funding was provided by the Portuguese Agency Fundação para a Ciência e a Tecnologia (SFRH/BPD/79542/2011 fellowship to SS and SFRH/BD/52225/2013 fellowship to JP), and by Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE): call H2020-MSCA-RISE-2014, Grant agreement 644167, 2015-2019.info:eu-repo/semantics/publishedVersio

Translocating the blood-brain barrier using electrostatics

Copyright © 2012 Ribeiro,Domingues, Freire,Santos and Castanho. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components—red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC).To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.Fundação para a Ciência e Tecnologia — Ministério da Educação e Ciência (FCT-MEC, Portugal) is acknowledged for funding (including fellowships SFRH/BD/42158/2007 to Marta M.B. Ribeiro, SFRH/BD/41750/2007 to Marco M. Domingues and SFRH/BD/70423/2010 to João M. Freire) and project PTDC/QUI-BIQ/119509/2010. Marie Curie Industry-Academia Partnerships and Pathways (European Commission) is also acknowledged for funding (FP7-PEOPLE-2007-3-1-IAPP, Project 230654)

Antibodies for the treatment of brain metastases, a dream or a reality?

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood-brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells.This research was funded by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP, grants PD/BD/128281/2017, PTDC/BBB-BQB/1693/2014 and PTDC/BBB-NAN/1578/2014.info:eu-repo/semantics/publishedVersio

HIV-1 Fusion Inhibitor Peptides Enfuvirtide and T-1249 Interact with Erythrocyte and Lymphocyte Membranes

Enfuvirtide and T-1249 are two HIV-1 fusion inhibitor peptides that bind to gp41 and prevent its fusogenic conformation, inhibiting viral entry into host cells. Previous studies established the relative preferences of these peptides for membrane model systems of defined lipid compositions. We aimed to understand the interaction of these peptides with the membranes of erythrocytes and peripheral blood mononuclear cells. The peptide behavior toward cell membranes was followed by di-8-ANEPPS fluorescence, a lipophilic probe sensitive to the changes in membrane dipole potential. We observed a fusion inhibitor concentration-dependent decrease on the membrane dipole potential. Quantitative analysis showed that T-1249 has an approximately eight-fold higher affinity towards cells, when compared with enfuvirtide. We also compared the binding towards di-8-ANEPPS labeled lipid vesicles that model cell membranes and obtained concordant results. We demonstrated the distinct enfuvirtide and T-1249 membranotropism for circulating blood cells, which can be translated to a feasible in vivo scenario. The enhanced interaction of T-1249 with cell membranes correlates with its higher efficacy, as it can increase and accelerate the drug binding to gp41 in its pre-fusion state