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© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ; The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio-orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei-infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 675671 (A.K. and G.J.L.B.). Funding from the Royal Society (URF\R\180019 to G.J.L.B.) and FCT Portugal (iFCT IF/00624/2015 to G.J.L.B.) is also acknowledged. ; info:eu-repo/semantics/publishedVersion

Micro- and nano-scale systems have emerged as important tools for developing clinically useful drug delivery systems. In this tutorial review, we discuss the exploitation of biomacromolecules for this purpose, focusing on proteins, polypeptides, nucleic acids and polysaccharides and mixtures thereof as potential building blocks for novel drug delivery systems. We focus on the mechanisms of formation of micro- and nano-scale protein-based capsules and shells, as well as on the functionalization of such structures for use in targeted delivery of bioactive materials. We summarise existing methods for protein-based capsule synthesis and functionalization and highlight future challenges and opportunities for delivery strategies based on biomacromolecules. ; U.S. is grateful to Professor Aharon Gedanken, Chemistry Department, Bar-Ilan University, Israel, for his support and supervision during her PhD research work. G.J.L.B. is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Lisboa. We thank Nuno Azoia assistance with the preparation of the figures. The authors thank the European Union Seventh Framework Programme (FP7/20072013) under grant agreement NMP4-LA-2009-228827 ...

© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ; We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement N° 836698 and under grant agreement N° 852985. Funding from the Scripps Research Institute (A.A.), FCT Portugal (PhD scholarship PD/BD/135512/2018 to C.F.A. and FCT Stimulus CEECIND/00453/2018 to G.J.L.B.) and Agencia Estatal Investigación of Spain (AEI; Grant RTI2018-099592-B-C21 to F.C.) is also acknowledged. ; info:eu-repo/semantics/publishedVersion

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, are reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly. ; SFRH/BD/81479/2011 and SFRH/BD/81269/2011 scholarships from Fundação para a Ciência e a Tecnologia (FCT). This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project ...

The incorporation of bioactive compounds in stealth nanoparticles or nanoemulsions enhances their half-life in systemic circulation and can overcome the problems associated with the free drug. Bovine serum albumin (BSA)-drug conjugates were produced with either methotrexate (MTX), a potent anticancer agent, or vancomycin (VCM), a potent antibiotic. Those conjugates were used to produce functionalized BSA nanoemulsions in a formulation composed by aqueous phase and organic phase. BSA-Folic acid (FA) conjugates were also produced allowing specific folate receptor (FR) mediated targeting of cancer cells (KB cell line). All conjugates had similar effects either in solution or in the form of nanoemulsions: BSA-MTX as anti-proliferative over Caco-2 cell line and BSA-VCM as lower minimum inhibitory concentration (MIC) comparatively to VCM solution on Staphylococcus aureus strain Newman. The production of nanoemulsions using BSA-drug conjugates for obtaining vectors loaded with stabilized drugs offers a good, flexible template for a wide range of medical applications. ; Ana Loureiro (SFRH/BD/81479/2011) holds a scholarship from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This work has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC - COMPETE and by Portuguese funds from FCT through the project ...

Copyright © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). ; Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A Neisseria meningitidis (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be O-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the O- and N-acetyl moieties through either H-bonding or CH–π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, while O-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies. ; This work was sponsored by GlaxoSmithKline Biologicals and has received funding from the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement 675671. ; info:eu-repo/semantics/publishedVersion

© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ; The emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains highlights the need to develop more efficacious and potent drugs. However, this goal is dependent on a comprehensive understanding of Mtb virulence protein effectors at the molecular level. Here, we used a post-expression cysteine (Cys)-to-dehydrolanine (Dha) chemical editing strategy to identify a water-mediated motif that modulates accessibility of the protein tyrosine phosphatase A (PtpA) catalytic pocket. Importantly, this water-mediated Cys-Cys non-covalent motif is also present in the phosphatase SptpA from Staphylococcus aureus, which suggests a potentially preserved structural feature among bacterial tyrosine phosphatases. The identification of this structural water provides insight into the known resistance of Mtb PtpA to the oxidative conditions that prevail within an infected host macrophage. This strategy could be applied to extend the understanding of the dynamics and function(s) of proteins in their native state and ultimately aid in the design of small-molecule modulators. ; We thank CNPq Brazil (fellowship 200456/2015-6 to J.B.B. and grants 454507/2014-3 and 300606/2010-9 to H.T.), the Fundação para a Ciência e a Tecnologia (FCT Investigator award IF/00624/2015 to G.J.L.B.), the European Union (Marie-Sklodowska Curie Innovative Training Network Protein Conjugates; Marie Skłodowska-Curie Individual Fellowship 743640 to T.R.; Marie-Curie Intra-European Fellowship 626890 to O.B.), the Ministerio de Economía, Industria, y Competitividad (project CTQ2015-67727-R to F.C.), and the Biotechnology and Biological Sciences Research Council (PhD studentship to L.D.) for funding. G.J.L.B. is a Royal Society University Research Fellow and the recipient of a European Research Council Starting Grant (TagIt, 676832). We also acknowledge funding by LISBOA-01-0145-FEDER-007391, co-financed ...

Bovine Serum Albumin (BSA) nanoemulsions were produced by high pressure homogenization with a tri-block copolymer (Poloxamer 407), which presents a central hydrophobic chain of polyoxypropylene (PPO) and two identical lateral hydrophilic chains of polyethylene glycol (PEG). We observed a linear correlation between tri-block copolymer concentration and size - the use of 5 mg/mL of Poloxamer 407 yields nanoemulsions smaller than 100 nm. Molecular dynamics and fluorescent tagging of the tri-block copolymer highlight their mechanistic role on the size of emulsions. This novel method enables the fabrication of highly stable albumin emulsions in the nano-size range, highly desirable for controlled drug delivery. Folic Acid (FA)-tagged protein nanoemulsions were shown to promote specific Folate Receptor (FR)-mediated targeting in FR positive cells. The novel strategy presented here enables the construction of size controlled, functionalized protein-based nanoemulsions with excellent characteristics for active targeting in cancer therapy. ; European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This work was supported by FEDER through POFC – COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014. The authors also thank the FCT Strategic Project of UID/BIO/04469/2013 ...

Copyright © 2022 The Authors. Published by American Chemical Society ; Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties. ; We thank the Agencia Estatal Investigación of Spain (AEI; Grants RTI2018-099592-B-C21 to F.C. and RTI2018-099592-B-C22 to G.J.-O.). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 675007. E.J.-M. thanks Universidad de la Rioja for ...

This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods. ; Financial support for this work was provided by the Marie Skłodow-ska-Curie Global Fellowships (749359-EnanSET, N.M.P) within the European Union research and inno-vation framework programme (2014-2020)

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor . These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance. ; Eugenia Nogueira (SFRH/BD/81269/2011), Ana Loureiro (SFRH/BD/81479/2011) and Catarina Almeida (SFRH/BPD/48533/2008) hold scholarships from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This study was also supported by FEDER through POFC-COMPETE, by national funds from FCT through the ...

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol. ; Eugenia Nogueira (SFRH/BD/81269/2011) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundacao para a Ciencia e a Tecnologia (FCT). Goncalo J. L. Bernardes is a Royal Society University Research Fellow at the Department of Chemistry, University of Cambridge and an Investigador FCT at the Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa. This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. This work was also supported by FCT I.P. through the strategic ...

This journal is © The Royal Society of Chemistry 2020. ; The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches. ; Funded under the Royal Society (URF to G. J. L. B., URF\R\180019), FCT Portugal (iFCT to G. J. L. B., IF/00624/2015, Postdoctoral Fellowship SFRH/BPD/95253/2013 to J. D. S., project PTDC/MED-QUI/28764/2017 to J. D. S., 02/SAICT/2017 grant 28333 to T. R., doctoral studentship SFRH/BD/143583/2019 to B. B. S., and DL 57/2016/CP1451/CT0025 to J. D. S.). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreements No. 807281 and 702428. The authors acknowledge the Wellcome Trust (104633/Z/14/Z, to D. W. and C. V. R.), Ministerio de Economía y Competitividad (project RTI2018-099592-B-C21 to F. C.), the Royal G. and Mae H. Westaway Family Memorial Fund (A. N. K.), National Science Foundation Graduate Research Fellowship (Grant No ...

© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ; Anti-obesity drugs in the amphetamine (AMPH) class act in the brain to reduce appetite and increase locomotion. They are also characterized by adverse cardiovascular effects with origin that, despite absence of any in vivo evidence, is attributed to a direct sympathomimetic action in the heart. Here, we show that the cardiac side effects of AMPH originate from the brain and can be circumvented by PEGylation (PEGyAMPH) to exclude its central action. PEGyAMPH does not enter the brain and facilitates SNS activity via theβ2-adrenoceptor, protecting mice against obesity by increasing lipolysis and thermogenesis, coupled to higher heat dissipation, which acts as an energy sink to increase energy expenditure without altering food intake or locomotor activity. Thus, we provide proof-of-principle for a novel class of exclusively peripheral anti-obesity sympathofacilitators that are devoid of any cardiovascular and brain-related side effects. ; This work was supported by the Fundação Para a Ciência e Tecnologia (FCT - PTDC-BIM-MET-3750-2014 and LISBOA-01-0145-FEDER-030892), the European Molecular Biology Organization (EMBO - Installation Grant 3037), the Human Frontier Science Program (HFSP - RGY0070/2016), Maratona da Saúde (Diabetes - 2016), the European Research Council (ERC-2017-COG-771431), and the Howard Hughes Medical Institute/Wellcome International research scholar award (HHMI - 208576/Z/17/Z). G.J.L.B. is a Royal Society university Research Fellow (URF\R\180019) and FCT Investigator (IF/00624/2015). B.J. and A.K. were supported by the BBSRC (BB/M027252/1/bbsrc). We also acknowledge the Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-022125), the Project EU_FT-ICR_MS, funded by the European Union Horizon 2020 research and innovation program under grant agreement no. 731077. The contribution of Y.S., M.O., and F.C. was supported by Ministerio de Ciencia, ...