Examining Topoisomers of a Snake-Venom-Derived Peptide for Improved Antimicrobial and Antitumoral Properties

Ctn[15-34], the C-terminal section of crotalicidin (Ctn), a cathelicidin from a South American pit viper, is an antimicrobial and antitumoral peptide with remarkably longer stability in human serum than the parent Ctn. In this work, a set of topoisomers of both Ctn and Ctn[15-34], including the retro, enantio, and retroenantio versions, were synthesized and tested to investigate the structural requirements for activity. All topoisomers were as active as the cognate sequences against Gram-negative bacteria and tumor cells while slightly more toxic towards normal cells. More importantly, the enhanced serum stability of the D-amino-acid-containing versions suggests that such topoisomers must be preferentially considered as future antimicrobial and anticancer peptide leads

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

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.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.This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grants SAF2011-24899 and AGL2014-52395-C2, by Fundação para a Ciência e a Tecnologia (FCT, Portugal) Grants PTDC/QEQ-MED/4412/2014, and by EU Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program Grant 644167, 2015–2019.info:eu-repo/semantics/publishedVersio

Teleneuropsicología: Experiencia del Instituto Neurológico de Colombia durante confinamiento obligatorio por covid-19, año 2020

Background: The absence of specific treatments for the COVID 19 has promoted the design of strategies in public health to reduce the transmission rate and decrease the mortality from the virus. Colombian government decreed a mandatory quarantine that started on march 24, 2020. This measure became an opportunity for the implementation of telehealth in order to continue the provision of health services. The Neuropsychological division of the Instituto Neurologico de Colombia (INDEC), decided to establish a neuropsychology patient care service using telehealth and telecare. Methods: this article will set out the details of this experience considering and describing administrative, informatics, clinical, logistical, ethical and legal aspects. Results and Conclusions: this modality of work, in a neuropsychological unit, reveals that is possible to capitalize on the maturity that has been reached in the clinical, technological and administrative field to created fast transformations that respond accordingly to the actual needs.Introducción: la ausencia de tratamientos específicos para COVID-19 ha promovido el diseño de estrategias en salud pública para reducir tasas de contagio y disminuir la mortalidad por el virus. En Colombia se decretó una cuarentena obligatoria a partir del 24 de marzo de 2020. Esta medida se convirtió en una oportunidad para la implementación de programas de consulta mediante telesalud, con el fin de continuar la prestación de servicios de salud. La Unidad de Neuropsicología del Instituto Neurológico de Colombia (INDEC) decidió instaurar un modelo de atención en neuropsicología mediante la metodología de telesalud. Métodos: en el presente artículo se exponen los detalles de esta experiencia, considerando y describiendo los aspectos administrativos, informáticos, clínicos, logísticos, éticos y legales. Resultados y conclusiones: esta modalidad de trabajo de una unidad de neuropsicología, revela que es posible capitalizar la madurez que se ha alcanzado en el campo clínico, tecnológico y administrativo, para generar transformaciones de forma rápida y que respondan de manera oportuna a las necesidades del medio

Hitchhiking with Nature : exploring snake venom peptides to fight cancer and superbugs

This work describes the discovery and optimization of efficient, selective, cost-effective and proteolytic-resistant antimicrobial and antitumoral peptides. To this end, we first identified novel cathelicidin-related peptides from the venom gland of South American pit vipers, collectively named vipericidins. In particular, crotalicidin (Ctn), a 34-mer helical peptide from the Crotalus durissus terrificus venom, highlighted due to its promising antimicrobial and antitumoral properties. Its fragment Ctn[15-34] also stood out due to its overall preserved activity, improved selectivity, serum stability and significant size reduction. Their mechanisms of action were stepwise characterized against Escherichia coli and a leukemic cell line. The unusually high lifespan of Ctn[15-34] in serum was also investigated to ascertain its structural determinants and molecular interaction with serum proteins. Altogether, this thesis proposes two novel bioactive peptides as potential drug candidates to fight bacterial infections and cancer, particularly leukemia. Beyond specific results, this thesis provides a set of valuable methodologies to discover, optimize and evaluate bioactive molecules from natural sources.Esta tesis aborda el descubrimiento y optimización de péptidos antimicrobianos y antitumorales eficientes, selectivos, rentables y resistentes a la proteólisis. Con este fin, inicialmente identificamos nuevas catelicidinas en la glándula venenosa de diferentes víboras de América del Sur, denominadas vipericidinas. En particular la crotalicidina (Ctn), un péptido helicoidal de 34 residuos aislado del veneno de la serpiente Crotalus durissus terrificus destacó por sus propiedades antimicrobianas y antitumorales, así como su fragmento Ctn[15-34], que mostró una selectividad y estabilidad en suero mejoradas y una reducción de tamaño considerable. Los mecanismos de acción de ambos péptidos contra Escherichia coli y una línea celular de leucemia fueron investigados. También se investigó en profundidad la inusual estabilidad en suero de Ctn[15-34], con el fin de conocer sus determinantes estructurales y las posibles interacciones con proteínas del suero. En conjunto, esta tesis propone dos nuevos péptidos bioactivos como posibles candidatos para combatir infecciones bacterianas y la leucemia, además de proporcionar un conjunto de metodologías útiles en el descubrimiento, optimización y caracterización de moléculas bioactivas a partir de fuentes naturales

Hitchhiking with nature: snake venom peptides to fight cancer and superbugs

For decades, natural products in general and snake venoms (SV) in particular have been a rich source of bioactive compounds for drug discovery, and they remain a promising substrate for therapeutic development. Currently, a handful of SV-based drugs for diagnosis and treatment of various cardiovascular disorders and blood abnormalities are on the market. Likewise, far more SV compounds and their mimetics are under investigation today for diverse therapeutic applications, including antibiotic-resistant bacteria and cancer. In this review, we analyze the state of the art regarding SV-derived compounds with therapeutic potential, focusing on the development of antimicrobial and anticancer drugs. Specifically, information about SV peptides experimentally validated or predicted to act as antimicrobial and anticancer peptides (AMPs and ACPs, respectively) has been collected and analyzed. Their principal activities both in vitro and in vivo, structures, mechanisms of action, and attempts at sequence optimization are discussed in order to highlight their potential as drug leads

The use of a selective, nontoxic dual-acting peptide for breast cancer patients with brain metastasis

© 2024 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of commonly targeted receptors. Unspecific chemotherapy is currently the main therapeutic option, with poor results. Another major challenge is the frequent appearance of brain metastasis (BM) associated with a significant decrease in patient overall survival. The treatment of BM is even more challenging due to the presence of the blood-brain barrier (BBB). Here, we present a dual-acting peptide (PepH3-vCPP2319) designed to tackle TNBC/BM, in which a TNBC-specific anticancer peptide (ACP) motif (vCPP2319) is joined to a BBB peptide shuttle (BBBpS) motif (PepH3). PepH3-vCPP2319 demonstrated selectivity and efficiency in eliminating TNBC both in monolayers (IC50≈5.0 µM) and in spheroids (IC50≈25.0 µM), with no stringent toxicity toward noncancerous cell lines and red blood cells (RBCs). PepH3-vCPP2319 was also able to cross the BBB in vitro and penetrate the brain in vivo, and was stable in serum with a half-life above 120 min. Tumor cell-peptide interaction is fast, with quick peptide internalization via clathrin-mediated endocytosis without membrane disruption. Upon internalization, the peptide is detected in the nucleus and the cytoplasm, indicating a multi-targeted mechanism of action that ultimately induces irreversible cell damage and apoptosis. In conclusion, we have designed a dual-acting peptide capable of brain penetration and TNBC cell elimination, thus expanding the drug arsenal to fight this BC subtype and its BM.The project leading to these results have received funding from 'la Caixa' Foundation (ID 100010434) and FCT, I.P under the agreement LCF/PR/HP21/52310015; and “Fundação para a Ciência e Tecnologia” (FCT, Portugal) (grant: PD/BD/128281/2017, PTDC/BTM-MAT/2472/2021, UIDB/04349/2020, PTDC/QUI-OUT/3854/2021, and 2021.00895.CEECIND). Work at UPF Department of Medicine and Life Sciences (MELIS) also supported by MCIN “María de Maeztu” Program for Units of Excellence in R&D (CEX2018–000792-M).info:eu-repo/semantics/publishedVersio

Structural determinants conferring unusual long life in human serum to rattlesnake‐derived antimicrobial peptide Ctn(15‐34)

© 2019 European Peptide Society and John Wiley & Sons, Ltd.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.This work was supported by grants SAF2011-24899, AGL2014-52395-C2 and AGL2017-84097-C2-2-R from the Spanish Ministry of Economy and Competitiveness (MINECO), by Fundação para a Ciência e a Tecnologia (FCT, Portugal) grant PTDC/QEQ-MED/4412/2014, and by the EU Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program grant 644167, 2015-2019. C.P.-P. acknowledges the MINECO “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0370) for a predoctoral fellowship. S.A.D., D.A.M., and A.S.V. also acknowledge FCT for fellowships PD/BD/114425/2016, PD/BD/136752/2018, and funding under the Investigator Program IF/00803/2012.info:eu-repo/semantics/publishedVersio

Examining topoisomers of a snake-venom-derived peptide for improved antimicrobial and antitumoral properties

Ctn[15-34], the C-terminal section of crotalicidin (Ctn), a cathelicidin from a South American pit viper, is an antimicrobial and antitumoral peptide with remarkably longer stability in human serum than the parent Ctn. In this work, a set of topoisomers of both Ctn and Ctn[15-34], including the retro, enantio, and retroenantio versions, were synthesized and tested to investigate the structural requirements for activity. All topoisomers were as active as the cognate sequences against Gram-negative bacteria and tumor cells while slightly more toxic towards normal cells. More importantly, the enhanced serum stability of the D-amino-acid-containing versions suggests that such topoisomers must be preferentially considered as future antimicrobial and anticancer peptide leads.Financial support from the Spanish Ministry of Economy and Innovation (grant AGL2017-84097-C2-2-R), from the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033 grants PID2020-113184RB-C22; PID2020-112821GB-I00 and PID2020-114627RB-I00; MCIN/AEI/10.13039/501100011033/FEDER PDC2021-121544-I00), from the EU (Next GenerationEU/PRTR) and from La Caixa Banking Foundation (HR17-00409) is acknowledged

To what extent do fluorophores bias the biological activity of peptides? A practical approach using membrane-active peptides as models

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