Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• Keywords
• Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• COVID-19 virology   MeSH
• COVID-19 Drug Treatment * MeSH
• HEK293 Cells MeSH
• Protease Inhibitors * pharmacology   chemistry   MeSH
• Coronavirus 3C Proteases * metabolism   genetics   antagonists & inhibitors   chemistry   MeSH
• Humans MeSH
• Mutation MeSH
• Drug Discovery * MeSH
• SARS-CoV-2 * enzymology   drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 3C-like proteinase, SARS-CoV-2 MeSH Browser
• Antiviral Agents * MeSH
• Protease Inhibitors * MeSH
• Coronavirus 3C Proteases * 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 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.

• Keywords
• HPMA copolymer, cancer, fluorescence, iBody, imaging, protease,
• MeSH
• Acrylamides chemistry   MeSH
• Fluorescent Dyes * chemistry   chemical synthesis   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Neoplasms * diagnostic imaging   MeSH
• Optical Imaging methods   MeSH
• Polymers chemistry   MeSH
• Peptide Hydrolases metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Acrylamides MeSH
• Fluorescent Dyes * MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Browser
• Polymers MeSH
• Peptide Hydrolases MeSH

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

• Keywords
• HPMA copolymer, PD-1, PD-L1, T-cell, antibody mimetic, immune checkpoint, immunosuppression, immunotherapy, inhibitor, tumor immunology,
• MeSH
• B7-H1 Antigen * antagonists & inhibitors   immunology   metabolism   MeSH
• Immune Checkpoint Inhibitors * pharmacology   chemistry   MeSH
• Humans MeSH
• Antibodies, Monoclonal chemistry   pharmacology   MeSH
• Cell Line, Tumor MeSH
• Polymers chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• B7-H1 Antigen * MeSH
• CD274 protein, human MeSH Browser
• Immune Checkpoint Inhibitors * MeSH
• Antibodies, Monoclonal MeSH
• Polymers 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.

• Keywords
• HPMA copolymer, cancer, fluorescence, iBody, imaging, protease,
• Publication type
• Journal Article MeSH
• Preprint MeSH

Aqueous solutions of some polymers exhibit a lower critical solution temperature (LCST); that is, they form phase-separated aggregates when heated above a threshold temperature. Such polymers found many promising (bio)medical applications, including in situ thermogelling with controlled drug release, polymer-supported radiotherapy (brachytherapy), immunotherapy, and wound dressing, among others. Yet, despite the extensive research on medicinal applications of thermoresponsive polymers, their biodistribution and fate after administration remained unknown. Thus, herein, they studied the pharmacokinetics of four different thermoresponsive polyacrylamides after intramuscular administration in mice. In vivo, these thermoresponsive polymers formed depots that subsequently dissolved with a two-phase kinetics (depot maturation, slow redissolution) with half-lives 2 weeks to 5 months, as depot vitrification prolonged their half-lives. Additionally, the decrease of TCP of a polymer solution increased the density of the intramuscular depot. Moreover, they detected secondary polymer depots in the kidneys and liver; these secondary depots also followed two-phase kinetics (depot maturation and slow dissolution), with half-lives 8 to 38 days (kidneys) and 15 to 22 days (liver). Overall, these findings may be used to tailor the properties of thermoresponsive polymers to meet the demands of their medicinal applications. Their methods may become a benchmark for future studies of polymer biodistribution.

• Keywords
• LCST, biodistribution, poly(2,2-difluoroethyl)acrylamide, poly(N,N-diethylacrylamide), poly(N-acryloylpyrolidine), poly(N-isopropylacrylamide), polyacrylamide, rational polymer design,
• MeSH
• Mice MeSH
• Polymers * MeSH
• Temperature MeSH
• Tissue Distribution MeSH
• Drug Liberation MeSH
• Water * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Polymers * MeSH
• Water * 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.

• Keywords
• HPMA copolymer, PSMA, antibody mimetics, bicyclic phage display, chemical biology, cyclic peptide, molecular recognition, nanotechnology, phage display, protein targeting,
• MeSH
• Biomimetic Materials chemistry   MeSH
• Kallikreins chemistry   MeSH
• Humans MeSH
• Drug Carriers chemistry   MeSH
• Peptide Library * MeSH
• Prostate-Specific Antigen chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kallikreins MeSH
• KLK3 protein, human MeSH Browser
• Drug Carriers MeSH
• Peptide Library * MeSH
• Prostate-Specific Antigen MeSH

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

• MeSH
• Insulin-Like Growth Factor I metabolism   MeSH
• Insulin-Like Growth Factor II metabolism   MeSH
• Binding, Competitive MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Iodine Radioisotopes MeSH
• Radioligand Assay methods   MeSH
• Receptor, IGF Type 2 immunology   ultrastructure   MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IGF1 protein, human MeSH Browser
• IGF2 protein, human MeSH Browser
• IGF2R protein, human MeSH Browser
• Insulin-Like Growth Factor I MeSH
• Insulin-Like Growth Factor II MeSH
• Iodine-125 MeSH Browser
• Iodine Radioisotopes MeSH
• Receptor, IGF Type 2 MeSH

BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

• Keywords
• Blood digestion, Cathepsin, Cysteine peptidase, Diplozoidae, Eudiplozoon nipponicum, Fish parasite, Haematophagy, Monogenea, Protease, S2 subsite,
• MeSH
• Gastrointestinal Tract parasitology   MeSH
• Hydrolysis MeSH
• Host-Parasite Interactions MeSH
• Carps parasitology   MeSH
• Cathepsin B chemistry   genetics   isolation & purification   metabolism   MeSH
• Cathepsin L chemistry   genetics   isolation & purification   metabolism   MeSH
• Proteolysis MeSH
• Recombinant Proteins analysis   genetics   isolation & purification   MeSH
• Gene Expression Profiling MeSH
• Trematoda enzymology   genetics   MeSH
• Introduced Species MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cathepsin B MeSH
• Cathepsin L MeSH
• Recombinant Proteins MeSH

The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.

• MeSH
• Circular Dichroism MeSH
• Databases, Protein MeSH
• Datasets as Topic MeSH
• Models, Molecular * MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Peptide Library * MeSH
• Protein Aggregates MeSH
• Recombinant Proteins chemistry   isolation & purification   toxicity   MeSH
• Solubility MeSH
• Protein Folding MeSH
• Protein Structure, Secondary * MeSH
• Amino Acid Sequence MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Peptide Library * MeSH
• Protein Aggregates MeSH
• Recombinant Proteins MeSH

Here, we describe a novel polymer platform suitable for efficient diagnostics and potential theranostics based on 89Zr-labeled N-(2-hydroxypropyl)methacrylamide (HPMA)-based copolymer conjugates. A set of polymers differing in molecular weight with either low dispersity or high dispersity were designed and synthesized and their biodistribution in vivo was successfully and precisely observed over 72 h. Moreover, the feasibility of two imaging techniques, fluorescence imaging (FI) and positron emission tomography (PET), was compared using labeled polymer conjugates. Both methods gave comparable results thus showing the enhanced diagnostic potential of the prepared polymer-dye or polymer-chelator-89Zr constructs. The in vivo and ex vivo PET/FI studies indicated that the dispersity and molecular weight of the linear HPMA polymers have a significant influence on the pharmacokinetics of the polymer conjugates. The higher molecular weight and narrower distribution of molecular weights of the polymer carriers improve their pharmacokinetic profile for highly prolonged blood circulation and enhanced tumor uptake. Moreover, the same polymer carrier with the anticancer drug doxorubicin bound by a pH-sensitive hydrazone bond showed higher cytotoxicity and cellular uptake in vitro. Therefore, HPMA copolymers with low dispersity and a molecular weight near the limit of renal filtration can be used as highly efficient polymer carriers of tumor-targeted therapeutics or for theranostics with minimal side effects.

• MeSH
• Doxorubicin administration & dosage   pharmacokinetics   MeSH
• Neoplasms, Experimental diagnostic imaging   drug therapy   MeSH
• Jurkat Cells MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Drug Carriers chemistry   MeSH
• Optical Imaging * MeSH
• Polymers chemistry   MeSH
• Positron-Emission Tomography * MeSH
• Radioisotopes MeSH
• Theranostic Nanomedicine * MeSH
• Tissue Distribution MeSH
• Zirconium MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Doxorubicin MeSH
• Drug Carriers MeSH
• Polymers MeSH
• Radioisotopes MeSH
• Zirconium-89 MeSH Browser
• Zirconium MeSH