pH-Triggered Reversible Multiple Protein-Polymer Conjugation Based on Molecular Recognition

Polymer conjugation for protein-based therapeutics has been developed extensively, but it still suffers from conjugation leading to decrease in protein activity and generates complexes with limited diversity due to general classical systems only incorporating one protein per each complex. Here we introduce a site-specific noncovalent protein-polymer conjugation, which can reduce the heterogeneity of the conjugates without disrupting protein function, while allowing for the modulation of binding affinity and stability, affecting the pH dependent binding of the number of proteins per polymer. We compared classical one protein-polymer conjugates with multiple protein-polymer conjugates using His-tagged enhanced yellow fluorescence protein (His6-eYFP) and metal-coordinated tris-nitrilotriacetic acid (trisNTA-Me(n+)) in a site-specific way. trisNTA-Me(n+)-His6 acts as a reversible linker with pH-triggered release of functional protein from the trisNTA-functionalized copolymers. The nature of the selected Me(n+) and number of available trisNTA-Me(n+) on poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) (PNTn) copolymers enables predictable modulation of the conjugates binding affinity (0.09-1.35 μM), stability, cell toxicity, and pH responsiveness. This represents a promising platform that allows direct control over the properties of multiple protein-polymer conjugates compared to the classical single protein-polymer conjugates

Stimuli-Responsive Codelivery of Oligonucleotides and Drugs by Self-Assembled Peptide Nanoparticles

Ever more emerging combined treatments exploiting synergistic effects of drug combinations demand smart, responsive codelivery carriers to reveal their full potential. In this study, a multifunctional stimuli-responsive amphiphilic peptide was designed and synthesized to self-assemble into nanoparticles capable of co-bearing and -releasing hydrophobic drugs and antisense oligonucleotides for combined therapies. The rational design was based on a hydrophobic l-tryptophan-d-leucine repeating unit derived from a truncated sequence of gramicidin A (gT), to entrap hydrophobic cargo, which is combined with a hydrophilic moiety of histidines to provide electrostatic affinity to nucleotides. Stimuli-responsiveness was implemented by linking the hydrophobic and hydrophilic sequence through an artificial amino acid bearing a disulfide functional group (H3SSgT). Stimuli-responsive peptides self-assembled in spherical nanoparticles in sizes (100–200 nm) generally considered as preferable for drug delivery applications. Responsive peptide nanoparticles revealed notable nucleotide condensing abilities while maintaining the ability to load hydrophobic cargo. The disulfide cleavage site introduced in the peptide sequence induced responsiveness to physiological concentrations of reducing agent, serving to release the incorporated molecules. Furthermore, the peptide nanoparticles, singly loaded or coloaded with boron-dipyrromethene (BODIPY) and/or antisense oligonucleotides, were efficiently taken up by cells. Such amphiphilic peptides that led to noncytotoxic, reduction-responsive nanoparticles capable of codelivering hydrophobic and nucleic acid payloads simultaneously provide potential toward combined treatment strategies to exploit synergistic effects

DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles

Self-organization of nanocomponents was mainly focused on solid nanoparticles, quantum dots, or liposomes to generate complex architectures with specific properties, but intrinsically limited or not developed enough, to mimic sophisticated structures with biological functions in cells. Here, we present a biomimetic strategy to self-organize synthetic nanocompartments (polymersomes) into clusters with controlled properties and topology by exploiting DNA hybridization to interconnect polymersomes. Molecular and external factors affecting the self-organization served to design clusters mimicking the connection of natural organelles: fine-tune of the distance between tethered polymersomes, different topologies, no fusion of clustered polymersomes, and no aggregation. Unexpected, extended DNA bridges that result from migration of the DNA strands inside the thick polymer membrane (about 12 nm) represent a key stability and control factor, not yet exploited for other synthetic nano-object networks. The replacement of the empty polymersomes with artificial organelles, already reported for single polymersome architecture, will provide an excellent platform for the development of artificial systems mimicking natural organelles or cells and represents a fundamental step in the engineering of molecular factories

Site-selective template-directed synthesis of antibody Fc conjugates with concomitant ligand release

Template-directed methods are emerging as some of the most effective means to conjugate payloads at selective sites of monoclonal antibodies (mAbs). We have previously reported a method based on an engineered Fc-III reactive peptide to conjugate a radionuclide chelator to K317 of antibodies with the concomitant release of the Fc-III peptide ligand. Here, our method was redesigned to target two lysines proximal to the Fc-III binding site, K248 and K439. Using energy minimization predictions and a semi-combinatorial synthesis approach, we sampled multiple Fc-III amino acid substituents of A3, H5, L6 and E8, which were then converted into Fc-III reactive conjugates. Middle-down MS/MS subunit analysis of the resulting trastuzumab conjugates revealed that K248 and K439 can be selectively targeted using the Fc-III reactive variants L6Dap, L6Orn, L6Y and A3K or A3hK, respectively. Across all variants tested, L6Orn-carbonate appeared to be the best candidate, yielding a degree and yield of conjugation of almost 2 and 100% for a broad array of payloads including radionuclide chelators, fluorescent dyes, click-chemistry reagents, pre-targeted imaging reagents, and some cytotoxic small molecules. Furthermore, L6Orn carbonate appeared to yield similar conjugation results across multiple IgG subtypes. In vivo proof of concept was achieved by conjugation of NODAGA to the PD1/PD-L1 immune checkpoint inhibitor antibody atezolizumab, followed by PET imaging of PD-L1 expression in mice bearing PD-L1 expressing tumor xenograft using radiolabeled [64Cu]Cu-atezolizumab.|A one step template-directed method for site-specific conjugation of payloads to monoclonal antibodies is reported. Near 100% modification at a single lysine residue of the antibody Fc domain is achieved with a drug to antibody ratio of 2.ISIC-MSEA

Org Biomol Chem

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-beta-alanine () was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms

Fluorescent amino acids as versatile building blocks for chemical biology

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein–protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging. [Figure not available: see fulltext.]

Fluorophores ratiométriques pour le marquage de peptides et d'oligonucléotides‎ : applications à la protéine de la nucléocapside de VIH-1‎

Ce travail porte sur le développement de sondes fluorescentes 3-hydroxychromones pour suivre l’interaction de protéines avec des oligonucléotides (ODNs) et des membranes. Ces sondes ont deux bandes d'émission bien séparées et sensibles à l'environnement, résultant d’un transfert de proton intramoléculaire à l’état excité, d’où une détection des interactions via leur rapport d'intensité. Un analogue d’acide aminé dérivé de la sonde 3HC a été synthétisé puis inséré à des positions ciblées de la protéine de la nucléocapside (NC) du VIH-1. Nous avons ainsi obtenus des informations site-spécifique sur les modifications induites à proximité du site marqué lors de l’interaction avec des ODNs, une alternative étant d’utiliser des nucléosides fluorescents introduits en différentes positions des ODNs. Pour étudier les interactions peptide-membrane, une sonde 3HC sensible aux changements de polarité en solvants apolaires a été couplée à l’extrémité N-terminale de peptides synthétiques (mélittine, magaïnine, poly-L-lysine) connus pour interagir avec les biomembranes. Le rapport d'intensité se corréle bien avec la profondeur d'insertion de cette région N-terminale. Enfin, nous avons appliqué cette sonde pour détecter des interactions possibles de la NC avec les membranes. Nous avons montré que NC se lie avec une forte affinité par voie électrostatique aux membranes contenant des lipides anioniques, permettant de localiser NC au niveau des têtes lipidiques. NC déstabilise la membrane de manière dose-dépendante. Les complexes NC-ADN se lient également aux membranes comme la NC libre, permettant de suggérer une participation de NC à l'import nucléaire du complexe de pré-intégration.This work focuses on the development of a methodology for sensing interactions of proteins with oligonucleotides (ODNs) and membranes based on fluorescent probes from the 3-hydroxychromone (3HC) family. Due to an excited state intramolecular proton transfer, these dyes exhibit two highly resolved emission bands, differently sensitive to the environment, thus allowing to sense interactions through their intensity ratio changes. An amino acid analogue based on 3HC was synthesized and inserted at selected positions of the HIV-1nucleocapsid protein (NC), to further characterize the peptide-ODNs interaction and provide site-specific information on the environmental changes induced by the interaction close to the labeling site. As an alternative, fluorescent nucleosides were synthesized and applied to different positions of ODNs. To study the peptide-membrane interactions, 3HC probe sensitive to polarity changes in apolar solvents was coupled to the N-terminus of melittin, magainin and poly-L-lysine peptides, known to interact with lipid membranes. The observed intensity ratio of the probe correlates well with the insertion depth of the N-terminal region of the peptides. Finally, we applied this dye for the detection of possible interactions of NC with membranes. We have shown that NC binds with high affinity to membranes containing negatively charged lipids. This interaction is mainly driven by electrostatic forces and locates NC at the level of lipid heads. Moreover, NC destabilizes the membrane in a concentration-dependent manner. As free NC, NC-DNA complexes can also bind to membranes, suggesting an involvement of NC in the nuclear import of the pre-integration complex

Біопсія сигнальних лімфатичних вузлів: стан проблеми, доказова база та рекомендації (огляд літератури)

Актуальність. Концепція сигнальних лімфатичних вузлів (СЛВ) є активно досліджуваним і перспективним напрямком онкохірургії, що дозволяє індивідуалізувати підходи до лікування онкологічних хворих. Мета дослідження: огляд рекомендацій European Society of Medical Oncology (ESMO), National Comprehensive Cancer Network (NCCN) і публікацій у The Cochrane Library, що були присвячені питанню онкологічної ефективності біопсії СЛВ і традиційної лімфодисекції у лікуванні первинних солідних пухлин. Матеріали та методи. Розглянуто чинні рекомендації ESMO та NCCN, публікації в Cochrane Library, в яких у аспекті хірургічного лікування розглядається концепція СЛВ та її місце поруч із традиційним підходом до лімфодисекції. Результати. На сайті Кохранівської бібліотеки знайдено 3 огляди літератури, 7 рекомендацій ESMO та 8 — NCCN. Для ідентифікації СЛВ у шийній, аксилярній чи пахово­стегновій ділянці найбільшу інформативність мають комбіновані методи (барвник + радіофармпрепарат), у порожнині малого тазу та черевній порожнині — флуоресценти. Дослідження заморожених зрізів для меланоми шкіри не рекомендовано, при раку шийки матки — показано. Імуногістохімічне дослідження СЛВ є обов’язковим при раку статевого члена та меланомі, але недоцільним при раку молочної залози. Спільною точкою зору щодо онкологічних хворих залишається необхідність біопсії (тонкоголкової аспіраційної, трепан­біопсії чи ексцизійної) за підозри на метастатичне ураження лімфатичних вузлів чи при лімфаденопатії. Відсутність контрастованих СЛВ — показання до застосування традиційних підходів до лімфодисекції при відповідній онкологічній патології. Висновки. Розбіжності у висновках і рекомендаціях проаналізованої літератури пояснюються використанням як вихідних даних результатів різних досліджень. Рекомендації NCCN завдяки постійному динамічному оновленню містять найбільш актуалізовану інформацію. Враховуючи активний розвиток концепції СЛВ, накопичені за декілька років результати досліджень дозволяють обґрунтовано змінювати погляди на її місце у лікуванні онкологічних нозологій. Рекомендації ESMO та огляди Кохранівської спільноти, присвячені СЛВ, мають вже переважно історичне значення, і у більшості випадків їх застосування потребує критичного підходу з урахуванням результатів досліджень і метааналізів, що були опубліковані пізніше.</jats:p