MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1 beta and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3(L351P) knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants

HPMA Copolymer-Based Nanomedicines in Controlled Drug Delivery

Recently, numerous polymer materials have been employed as drug carrier systems in medicinal research, and their detailed properties have been thoroughly evaluated. Water-soluble polymer carriers play a significant role between these studied polymer systems as they are advantageously applied as carriers of low-molecular-weight drugs and compounds, e.g., cytostatic agents, anti-inflammatory drugs, antimicrobial molecules, or multidrug resistance inhibitors. Covalent attachment of carried molecules using a biodegradable spacer is strongly preferred, as such design ensures the controlled release of the drug in the place of a desired pharmacological effect in a reasonable time-dependent manner. Importantly, the synthetic polymer biomaterials based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers are recognized drug carriers with unique properties that nominate them among the most serious nanomedicines candidates for human clinical trials. This review focuses on advances in the development of HPMA copolymer-based nanomedicines within the passive and active targeting into the place of desired pharmacological effect, tumors, inflammation or bacterial infection sites. Specifically, this review highlights the safety issues of HPMA polymer-based drug carriers concerning the structure of nanomedicines. The main impact consists of the improvement of targeting ability, especially concerning the enhanced and permeability retention (EPR) effect

Highly Effective Synthetic Polymer-Based Blockers of Non-Specific Interactions in Immunochemical Analyses

In vitro diagnostic methods face non-specific interactions increasing their background level and influencing the efficacy and reproducibility. Currently, the most important and employed blocker of non-specific interactions is bovine serum albumin (BSA), an animal product with some disadvantages like its batch-to-batch variability and contamination with RNases. Herein, we developed amphiphilic water-soluble synthetic copolymers based on the highly biocompatible, non-immunogenic and nontoxic N-2-(hydroxypropyl)methacrylamide (HPMA)-based copolymers or poly(oxazoline)s as highly effective synthetic blockers of non-specific interactions and an effective BSA alternative. The highest blocking capacity was observed for HPMA-based polymers containing two hydrophobic anchors taking advantage of the combination of two structurally different hydrophobic molecules. Polymers prepared by free radical polymerisation with broader dispersity were slightly better in terms of surface covering. The sandwich ELISA evaluating human thyroid-stimulating Hormone in patient samples revealed that the designed polymers can fully replace BSA without compromising the assay results. Importantly, as a fully synthetic material, the developed polymers are fully animal pathogen-free; thus, they are highly important materials for further development

N -(2-Hydroxypropyl)methacrylamide-Based Linear, Diblock, and Starlike Polymer Drug Carriers: Advanced Process for Their Simple Production

International audienc

Micelle-Forming Block Copolymers Tailored for Inhibition of P-gp-Mediated Multidrug Resistance: Structure to Activity Relationship

Multidrug resistance (MDR) is often caused by the overexpression of efflux pumps, such as ABC transporters, in particular, P-glycoprotein (P-gp). Here, we investigate the di- and tri- block amphiphilic polymer systems based on polypropylene glycol (PPO) and copolymers of (N-(2-hydroxypropyl)methacrylamide) (PHPMA) as potential macromolecular inhibitors of P-gp, and concurrently, carriers of drugs, passively targeting solid tumors by the enhanced permeability and retention (EPR) effect. Interestingly, there were significant differences between the effects of di- and tri- block polymer-based micelles, with the former being significantly more thermodynamically stable and showing much higher P-gp inhibition ability. The presence of Boc-protected hydrazide groups or the Boc-deprotection method did not affect the physico-chemical or biological properties of the block copolymers. Moreover, diblock polymer micelles could be loaded with free PPO containing 5–40 wt % of free PPO, which showed increased P-gp inhibition in comparison to the unloaded micelles. Loaded polymer micelles containing more than 20 wt % free PPO showed a significant increase in toxicity; thus, loaded diblock polymer micelles containing 5–15 wt % free PPO are potential candidates for in vitro and in vivo application as potent MDR inhibitors and drug carriers

Tris-(Nitrilotriacetic Acid)-Decorated Polymer Conjugates as Tools for Immobilization and Visualization of His-Tagged Proteins

Recombinant proteins are commonly expressed with artificial affinity tags for purification, immobilization and characterization. The most frequently used tag, His-tag, is a sequence of consecutive histidine residues fused to the protein of interest. Specialized small molecules that bind His-tag are primarily used for purification, while antibodies are used for protein analysis. However, various issues may be encountered with the use of antibodies. Low inherent stability, the difficulty of introducing chemical modifications, and often-unreliable batch-to-batch consistency are among the limiting factors that call for better alternatives. Recently described polymer conjugates of N-(2-hydroxypropyl) methacrylamide and low-molecular-weight functional ligands, so-called iBodies, are antibody mimetics capable of replacing antibodies in biochemical applications. We tailored this system for methods utilizing His-tag by accessorizing the polymer carrier with tris-nitrilotriacetic acid targeting ligands. These anti-polyHis iBodies are additionally accessorized with fluorophores, enabling detection, and biotin ligands, enabling immobilization. Here, we characterized anti-polyHis iBodies and explored their use as antibody mimetics. We tested their stability, as well as the influence of different metal mediators and His-tag lengths on binding. With high affinity and stability, iBodies represent a new alternative for immobilization and visualization of His-tagged proteins

European energy security and the role of LNG

Thesis (M.A.)--Özyeğin University, Graduate School of Social Sciences, Department of Institute of Social Sciences Department of Public Law, 2017.İşbu çalışmanın amacı, hukuki açıdan Avrupa Birliği'nde enerji güvenliğinin nasıl algılandığı ve sıvılaştırılmış doğal gaz yani (dilimize geçtiği hali ile) LNG'nin bunu nasıl desteklediğinin açıklanmasıdır. Çalışma, enerjinin modern dünyadaki yerini, Avrupa'nın enerji güvenliği konseptini doğal gaz ve özellikle LNG kapsamında ne şekilde anladığı kapsamında değerlendirerek izah eder. Enerji güvenliğinin gerekliliği, hukuki bir bakış açısıyla ele alınmaktadır. Bahse konu konseptin hukuki açıdan anlaşılabilmesi, yalnızca hukukun tepkisiyle mümkündür ve Avrupa'nın enerji güvenliği konseptine bakış açısının analizine destek olmak için hukuki düzenlemeler ve somut vakalar örnek olarak kullanılmaktadır. Enerji güvenliğinin hukuki gelişiminin yanı sıra teknolojik gelişmeler de değerlendirilmekte ve bu açıdan LNG, enerji güvenliğine katkıları anlamında incelenmektedir. Çünkü, enerji olmaksızın ekonomi, hukuk olmaksızın enerji olmaz.The purpose of this work is to explain how energy security is perceived within the European Union and how liquified natural gas, in short LNG, supports it from legal aspect. This work states the place of energy in modern world by evaluating Europe's understanding of energy security concept under the scope of natural gas and especially LNG. The essentialness of energy security is discussed from a legal point of view. The aforesaid concept can only be understood by legal reactions and regulations and case studies are used as examples to support the analysis of Europe's energy security perception. Along with the legal development of energy security, technological development is also studied and LNG is discussed within the meaning of its reinforcement for the concept. Eventually, economy does not exist without energy; likewise energy does not exist without law

Structure-to-Efficacy Relationship of HPMA-Based Nanomedicines: The Tumor Spheroid Penetration Study

Nanomedicines are a novel class of therapeutics that benefit from the nano dimensions of the drug carrier. These nanosystems are highly advantageous mainly within cancer treatment due to their enhanced tumor accumulation. Monolayer tumor cells frequently used in routine preclinical assessment of nanotherapeutics do not have a spatial structural architecture that allows the investigation of the penetration of nanomedicines to predict their behavior in real tumor tissue. Therefore, tumor spheroids from colon carcinoma C26 cells and glioblastoma U87-MG cells were used as 3D in vitro models to analyze the effect of the inner structure, hydrodynamic size, dispersity, and biodegradability of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based nanomedicines carrying anticancer drug pirarubicin (THP) on the penetration within spheroids. While almost identical penetration through spheroids of linear and star-like copolymers and also their conjugates with THP was observed, THP penetration after nanomedicines application was considerably deeper than for the free THP, thus proving the benefit of polymer carriers. The cytotoxicity of THP-polymer nanomedicines against tumor cell spheroids was almost identical as for the free THP, whereas the 2D cell cytotoxicity of these nanomedicines is usually lower. The nanomedicines thus proved the enhanced efficacy within the more realistic 3D tumor cell spheroid system

HPMA Copolymer Mebendazole Conjugate Allows Systemic Administration and Possesses Antitumour Activity In Vivo

Mebendazole and other benzimidazole antihelmintics, such as albendazole, fenbendazole, or flubendazole, have been shown to possess antitumour activity, primarily due to their microtubule-disrupting activity. However, the extremely poor water-solubility of mebendazole and other benzimidazoles, resulting in very low bioavailability, is a serious drawback of this class of drugs. Thus, the investigation of their antitumour potential has been limited so far to administering repeated high doses given peroral (p.o.) or to using formulations, such as liposomes. Herein, we report a fully biocompatible, water-soluble, HPMA copolymer-based conjugate bearing mebendazole (P-MBZ; Mw 28–33 kDa) covalently attached through a biodegradable bond, enabling systemic administration. Such an approach not only dramatically improves mebendazole solubility but also significantly prolongs the half-life and ensures tumour accumulation via an enhanced permeation and retention (EPR) effect in vivo. This P-MBZ has remarkable cytostatic and cytotoxic activities in EL-4 T-cell lymphoma, LL2 lung carcinoma, and CT-26 colon carcinoma mouse cell lines in vitro, with corresponding IC50 values of 1.07, 1.51, and 0.814 µM, respectively. P-MBZ also demonstrated considerable antitumour activity in EL-4 tumour-bearing mice when administered intraperitoneal (i.p.), either as a single dose or using 3 intermittent doses. The combination of P-MBZ with immunotherapy based on complexes of IL-2 and anti-IL-2 mAb S4B6, potently stimulating activated and memory CD8+ T cells, as well as NK cells, further improved the therapeutic effect