Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

• Klíčová slova
• HPMA copolymer, cancer, fluorescence, iBody, imaging, protease,
• MeSH
• akrylamidy chemie   MeSH
• fluorescenční barviva * chemie   chemická syntéza   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory * diagnostické zobrazování   MeSH
• optické zobrazování metody   MeSH
• polymery chemie   MeSH
• proteasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• akrylamidy MeSH
• fluorescenční barviva * MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• polymery MeSH
• proteasy MeSH

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

• Klíčová slova
• HPMA copolymer, cancer, fluorescence, iBody, imaging, protease,
• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.

• Klíčová slova
• HPMA copolymer, PSMA, antibody mimetics, bicyclic phage display, chemical biology, cyclic peptide, molecular recognition, nanotechnology, phage display, protein targeting,
• MeSH
• biomimetické materiály chemie   MeSH
• kalikreiny chemie   MeSH
• lidé MeSH
• nosiče léků chemie   MeSH
• peptidová knihovna * MeSH
• prostatický specifický antigen chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kalikreiny MeSH
• KLK3 protein, human MeSH Prohlížeč
• nosiče léků MeSH
• peptidová knihovna * MeSH
• prostatický specifický antigen MeSH

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)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P 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.

• MeSH
• cytokiny antagonisté a inhibitory   MeSH
• furany farmakologie   MeSH
• HEK293 buňky MeSH
• heterocyklické sloučeniny tetra- a více cyklické MeSH
• indeny MeSH
• inflamasomy antagonisté a inhibitory   MeSH
• lidé MeSH
• lipopolysacharidy MeSH
• makrofágy účinky léků   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• periodické syndromy asociované s kryopyrinem genetika   MeSH
• preklinické hodnocení léčiv MeSH
• protein NLRP3 antagonisté a inhibitory   genetika   MeSH
• proteinové domény MeSH
• sulfonamidy farmakologie   MeSH
• sulfony MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• furany MeSH
• heterocyklické sloučeniny tetra- a více cyklické MeSH
• indeny MeSH
• inflamasomy MeSH
• lipopolysacharidy MeSH
• N-(1,2,3,5,6,7-hexahydro-S-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide MeSH Prohlížeč
• NLRP3 protein, human MeSH Prohlížeč
• Nlrp3 protein, mouse MeSH Prohlížeč
• protein NLRP3 MeSH
• sulfonamidy MeSH
• sulfony MeSH