10 research outputs found
Celebrating the 2022 Nobel Prize in Chemistry. How are click chemistry and bioorthogonal chemistry transforming biomedicine?
La quĂmica clic i la quĂmica bioortogonal han obert nous camins en la modificaciĂł de sistemes complexos i han contribuĂŻt a posar la quĂmica a l’abast de tothom. La quĂmica clic permet enllaçar molècules altament funcionalitzades d’una manera rĂ pida, senzilla i robusta, mentre que la quĂmica bioortogonal permet realitzar modificacions en entorns biològics. Tant una quĂmica com l’altra ofereixen moltes possibilitats i han obert nous horitzons en camps com la biomedicina, entre d’altres. Dins de la biomedicina, la quĂmica clic i la quĂmica bioortogonal permeten generar eines tant per a entendre fenòmens en cèl·lules i organismes com per a generar nous fĂ rmacs i estratègies terapèutiques.Click and bioorthogonal chemistry have opened new paths in the modification of complex systems and have contributed to making chemistry accessible to everyone. Click chemistry allows richly functionalized molecules to be linked quickly, easily and robustly, while bioorthogonal chemistry enables modifications to be made in biological environments. Both these branches of chemistry are highly versatile and have opened up new horizons in biomedicine, among other fields. Specifically, within the field of biomedicine, click and bioorthogonal chemistry permit the generation of tools both to understand phenomena in cells and organisms and to generate new drugs and therapeutic strategies
Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery
Brain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood-brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins
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High-efficacy subcellular micropatterning of proteins using fibrinogen anchors.
Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology
From bee venom to blood-brain barrier shuttles. Development of minimized apamin derivatives for brain delivery of antibodies and other cargoes
[spa] La mayor parte de fármacos diseñados para tratar enfermedades del sistema nervioso central no son efectivos porque no pueden cruzar la barrera hematoencefálica (BHE). Una de las estrategias más prometedoras para superar este obstáculo es el uso de lanzadoras peptĂdicas. Sin embargo, la eficiencia y selectividad de dichas lanzadoras aĂşn debe mejorar. Los objetivos principales de la tesis eran encontrar nuevas lanzadoras resistentes a proteases y aumentar el transporte de anticuerpos monoclonales a travĂ©s de la BHE. Teniendo en cuenta que la apamina es un pĂ©ptido del veneno de la abeja que cruza la BHE, iniciamos el estudio demostrando que los residuos implicados en la toxicidad no eran necesarios para el transporte. Luego generamos análogos simplificados y MiniAp-4 mostrĂł un mejor compromiso entre estabilidad en suero, permeabilidad y inmunogenicidad. Este pĂ©ptido incrementĂł el transporte de GFP, quantum dots y nanopartĂculas de oro en un modelo celular humano de BHE. A demás, aumentĂł en 7,6 veces la concentraciĂłn de cianina-5.5 en el cerebro de ratones. Por lo que se refiere al transporte de anticuerpos, pusimos a punto varios mĂ©todos de conjugaciĂłn de los pĂ©ptidos lanzadora a distintas partes de la inmunoglobulina, incluyendo extremos N-terminales, cadenas de oligosacáridos, lisinas y cisteĂnas. Ninguna conjugaciĂłn disminuyĂł la afinidad de los anticuerpos por sus epĂtopos exceptuando la del N-terminal. Escogimos la modificaciĂłn de cisteĂnas mediante quĂmica tiol-maleimida y tambiĂ©n la de lisinas utilizando cicloadiciĂłn alquino-azida catalizada por cobre (CuAAC) para generar conjugados usando varias lanzadoras. Algunos de ellos mostraron un transporte significativamente más elevado que el anticuerpo sin modificar en el modelo humano de BHE, en especial una mini-apamina y RVG29 enlazados mediante CuAAC. La mayor eficiencia de las lanzadoras unidas de este modo es atribuible a la mayor accesibilidad para interaccionar con sus receptores. En conclusiĂłn, en esta tesis hemos generado análogos de apamina resistentes a proteasas, más reducidos asĂ como menos tĂłxicos e inmunogĂ©nicos. Además, MiniAp-4 es capaz de transportar distintos compuestos a travĂ©s de la BHE in vitro e in vivo. TambiĂ©n hemos demostrado que el transporte de anticuerpos se puede aumentar mediante el uso de lanzadores peptĂdicas varias, incluyendo las mini-apaminas.[eng] Brain delivery is a major unmet challenge because most drugs cannot cross the blood-brain barrier (BBB). Despite the restrictive nature of this barrier, brain vasculature reaches essentially every neural cell to supply it with nutrients. BBB-shuttles are molecules that take advantage of endogenous transport mechanisms to deliver cargoes into the brain parenchyma. However, the efficiency and selectivity of current shuttles needs to be improved. It has recently been shown that one way to enhance their transport capacity is to make them protease-resistant. The main goals of this thesis were to find new protease-resistant BBB-shuttle peptides and also to increase the transport monoclonal antibodies across the barrier. A source of peptides with high resistance to proteases is venoms. Moreover, some components, such as the potassium channel blocker apamin, have an effect on the CNS without disrupting the BBB, which makes them good BBB-shuttle candidates. Firstly, we proved that apamin and the most similar analogue described not to be toxic had a similar permeability in a bovine cell-based BBB model, which indicated that the residues implied in toxicity were not required for the transport across the BBB. Then we generated a simplified analogue, MiniAp-1, which crossed the cell monolayer mainly through an active mechanism with higher permeability than apamin. This result encouraged us to prepare additional analogues and one of them, MiniAp-4, had a permeability 3-fold that of apamin. This increase in transport could be partly explained by its high resistance to serum proteases and its different conformational preferences with respect to the other analogues. We then demonstrated that MiniAp-4 significantly improved the transport of GFP, quantum dots and gold nanoparticles in a human cellbased model of the BBB. Finally, we showed that this peptide was capable of enhancing 7.6-fold the delivery of cyanine-5.5 in mice brain. Furthermore, we detected the targeted probe in the brain parenchyma. In parallel, we aimed to increase the transport of monoclonal antibodies against glioblastoma across the BBB. We set up five reactions to conjugate peptide shuttles to different parts of two antibodies. Modification of the transaminated N-termini through oxime ligation provided a peptide/antibody ratio (PAR) lower than 1. Conjugation of the fucose present in the glycan chains yielded a PAR of 2 with one antibody but was not applied to the other because of its complex glycosylation pattern. Conversely, we achieved a PAR of 4 on partially reduced interchain cystines of both antibodies. Hence, we used this method to generate a small library of BBB-shuttle-antibody conjugates. The thioether bond linking the shuttles to the antibodies proved stable at least for 24 h in human serum. We also linked peptides containing cysteines to the lysines using cuppercatalyzed azide-alkyne cycloaddition (CuAAC). No conjugation significantly reduced antibody affinity except for N-terminal ligation. When the library of conjugates was assayed in the human cell-based BBB model we observed a significant increase of the transport with most peptides. MiniAp-1 and RVG29 were the shuttles providing the highest enhancement. Regarding the modification site, conjugation to lysines using CuAAC was the most suitable, probably because shuttles are more accessible to their receptors. In conclusion, in this thesis we have shown that cyclic mini-apamins, with reduced toxicity and immunogenicity, are highly resistant to serum proteases and are capable of crossing a tight brain endothelial cell monolayer. MiniAp-4 efficiently delivers diverse cargoes across the BBB in a human cell-based model and in mice. We have also proved that some BBB-shuttles, particularly MiniAp-1 and RVG29, can enhance the transport of monoclonal antibodies in a cell-based model and that peptides linked to the lysines using CuAAC provide the highest increase.[cat] La major part de fĂ rmacs dissenyats per tractar malalties del sistema nerviĂłs central no sĂłn efectius perquè no poden creuar la barrera hematoencefĂ lica (BHE). Una de les estratègies mĂ©s prometedores per superar aquest obstacle Ă©s l’ús de llançadores peptĂdiques. Tanmateix, l’eficiència i selectivitat d’aquests encara s’ha de millorar. Els objectius principals de la tesi eren, per una banda, trobar noves llançadores resistents a proteases i, per l’altra, augmentar el transport d’anticossos monoclonals a travĂ©s de la BHE. Tenint en compte que l’apamina Ă©s un pèptid del verĂ de l’abella que creua la BHE, vam iniciar l’estudi demostrant que els residus implicats en la toxicitat no eren necessaris pel transport. Aleshores vam generar anĂ legs simplificats i MiniAp-4 va mostrar el millor compromĂs entre estabilitat en sèrum, permeabilitat i immunogenicitat. Aquest pèptid va incrementar el transport de la GFP, quantum dots i nanopartĂcules d’or en un model cel·lular humĂ de BHE. A mĂ©s, va augmentar en 7,6 vegades la concentraciĂł de cianina-5.5 en el cervell de ratolins. Pel que fa el transport d’anticossos, vam posar a punt diversos mètodes per conjugar els pèptids llançadora a diferents parts de la immunoglobulina, incloent els extrems Nterminals, les cadenes glicosĂdiques, les lisines i les cisteĂŻnes. Cap conjugaciĂł va disminuir l’afinitat dels anticossos pels seus epĂtops exceptuant la del N-terminal. Vam escollir la modificaciĂł de les cisteĂŻnes utilitzant quĂmica tiol-maleimida i tambĂ© la de les lisines mitjançant cicloaddiciĂł alquĂ-azida catalitzada per coure (CuAAC) per generar conjugats emprant diverses llançadores. Alguns d’aquests van mostrar un transport significativament mĂ©s elevat que l’anticòs sol en el model cel·lular humĂ de BHE, en especial una mini-apamina i el RVG29 enllaçats mitjançant CuAAC. La major eficiència de les llançadores amb aquesta uniĂł Ă©s atribuĂŻble a la major accessibilitat per interaccionar amb els seus receptors. Per concloure, en aquesta tesi hem generat anĂ legs d’apamina resistents a protaeses, mĂ©s reduĂŻts, menys tòxics i menys immunogènics. A mĂ©s, MiniAp-4 Ă©s capaç de transportar diversos compostos a travĂ©s de la BHE in vitro i in vivo. TambĂ© hem demostrat que el transport d’anticossos es pot augmentar mitjançant diferents llançadores peptĂdiques, incloent les mini-apamines
BrainBike peptidomimetic enables efficient transport of proteins across brain endothelium
International audienceProtein therapeutics cannot reach the brain in sufficient amounts because of their low permeability across the blood-brain barrier. Here we report a new family of bicyclic peptide shuttles, BrainBikes, capable of increasing transport of proteins, including antibody derivatives, in a human cell-based model of the blood-brain barrier
Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery
Brain delivery is one of the major challenges in drug development because of the high number of patients suffering from neural diseases and the low efficiency of the treatments available. Although the blood-brain barrier (BBB) prevents most drugs from reaching their targets, molecular vectors - known as BBB shuttles - offer great promise to safely overcome this formidable obstacle. In recent years, peptide shuttles have received growing attention because of their lower cost, reduced immunogenicity, and higher chemical versatility than traditional Trojan horse antibodies and other proteins
Protease-Resistant Peptides for Targeting and Intracellular Delivery of Therapeutics
Peptides show high promise in the targeting and intracellular delivery of next-generation bio- and nano-therapeutics. However, the proteolytic susceptibility of peptides is one of the major limitations of their activity in biological environments. Numerous strategies have been devised to chemically enhance the resistance of peptides to proteolysis, ranging from N- and C-termini protection to cyclization, and including backbone modification, incorporation of amino acids with non-canonical side chains and conjugation. Since conjugation of nanocarriers or other cargoes to peptides for targeting and cell penetration may already provide some degree of shielding, the question arises about the relevance of using protease-resistant sequences for these applications. Aiming to answer this question, here we provide a critical review on protease-resistant targeting peptides and cell-penetrating peptides (CPPs). Two main approaches have been used on these classes of peptides: enantio/retro-enantio isomerization and cyclization. On one hand, enantio/retro-enantio isomerization has been shown to provide a clear enhancement in peptide efficiency with respect to parent L-amino acid peptides, especially when applied to peptides for drug delivery to the brain. On the other hand, cyclization also clearly increases peptide transport capacity, although contribution from enhanced protease resistance or affinity is often not dissected. Overall, we conclude that although conjugation often offers some degree of protection to proteolysis in targeting peptides and CPPs, modification of peptide sequences to further enhance protease resistance can greatly increase homing and transport efficiency
Fortifier selection and dosage enables control of breast milk osmolarity.
BACKGROUND:Human breast milk (BM) fortification is required to feed preterm newborns with less than 32 weeks of gestation. However, addition of fortifiers increases osmolarity and osmolarity values higher than 450 mOsm/kg may be related to gastrointestinal pathology. Hence, fortifier selection and dosage are key to achieve optimal feeding. OBJECTIVES:To compare the effect on osmolality of adding different fortifications, including recently developed formulations, to BM and to study evolution of osmolarity over time in supplemented BM. METHODS:Frozen mature BM from 10 healthy mothers of premature newborns was fortified with each of the following human milk fortifiers (HMF): AlmirónFortifier®, NANFM85®, or PreNANFM85®. In addition, fortified BMs were modified with one of the following nutritional supplements (NS): Duocal MCT®, Nutricia® AminoAcids Mix, or Maxijul®. Osmolality of BM alone, fortified and/or supplemented was measured at 1 and 22 hours after their preparation. All samples were kept at 4°C throughout the study. RESULTS:Osmolality of BM alone was close to 300 mOsm/kg and did not change over 22 hours. When equicaloric amounts of HMF AlmirónFortifier®, NANFM85®, and PreNANFM85® were added to BM, osmolality increased roughly to 480 mOsm/kg with the first two fortifiers and only to 433±6 mOsm/kg with the third one. Upon addition of any of four different NSs to BM modified with AlmirónFortifier® and NANFM85®, osmolality reached values greater than 520 mOsm/kg, while osmolality of PreNANFM85® with two out of the four NSs remained below 490 mOsm/kg. NSs supplementing carbohydrates and hydrolysed proteins resulted into a higher increase of BM osmolarity. Osmolality increased significantly with time and, after 22h, only BM modified with PreNANFM85® remained below 450 mOsm/kg. CONCLUSIONS:Upon addition of the HMFs tested, BM osmolality increases significantly and keeps raising over time. All HMFs but the recently developed PreNAN FM85® at 4% exceed the AAP recommended threshold for osmolarity of 450 mOsm/kg. Addition of NSs to PreNAN FM85® at 4% significantly increases osmolality above 450 mOsm/Kg. Thus, using PreNAN FM85® at 5% may be preferable to adding nutritional supplements since nutritional recommendations by the ESPGHAN are reached with a lower increase in osmolality
MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery
Drug delivery across the blood-brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non-invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam-bridged peptidomimetic MiniAp-4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human-cell-based BBB model. Furthermore, MiniAp-4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice