The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

• Klíčová slova
• AlphaScreen, Cap snatching, DNA-linked Inhibitor Antibody assay, Influenza A polymerase, Inhibitor, Pimodivir,
• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• fluorometrie metody   MeSH
• lidé MeSH
• piperidiny farmakologie   MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• pyrroly MeSH
• RNA čepičky metabolismus   MeSH
• RNA-dependentní RNA-polymerasa antagonisté a inhibitory   metabolismus   MeSH
• rychlé screeningové testy * metody   MeSH
• virové proteiny * antagonisté a inhibitory   metabolismus   MeSH
• virus chřipky A účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• PB2 protein, Influenzavirus A MeSH Prohlížeč
• pimodivir MeSH Prohlížeč
• piperidiny MeSH
• pyridiny MeSH
• pyrimidiny MeSH
• pyrroly MeSH
• RNA čepičky MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové proteiny * MeSH

The influenza RNA-dependent RNA polymerase harbours an endonuclease subunit characterized by a catalytic site housing two divalent metal ions. By effectively chelating both Mg2+ and Mn2+ ions, a small-molecule inhibitor with a metal-binding pharmacophore can halt endonuclease activity. Herein, two 3'-dehydroxypurpurogallin-4-carboxamide series, namely twelve C-4' unsubstituted and twelve C-4' phenyl substituted congeners were designed and prepared to be tested as inhibitors of the metal-dependent viral enzyme. These inhibitors were accessed through the chemoenzymatic reaction of gallic acid with either pyrocatechol or phenylpyrocatechol moderated by laccase, followed by amidation. Experimental IC50 values were determined using AlphaScreen technology, with the most potent inhibitors exhibiting IC50 values around 0.35 μM. Using X-ray crystallography, we analyzed structure of the endonuclease in complex with one potent 3'-dehydroxypurpurogallin-carboxamide at 2.0 Å resolution, revealing the coordination of the compound's triad of oxygen atoms with the two metal ions in the influenza A endonuclease active site.

• Klíčová slova
• Chemical synthesis, Endonuclease, Enzyme inhibition, Influenza, Metalloenzymes,
• MeSH
• amidy chemie   farmakologie   chemická syntéza   MeSH
• antivirové látky * farmakologie   chemická syntéza   chemie   MeSH
• endonukleasy * antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů * chemie   chemická syntéza   farmakologie   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• mikrobiální testy citlivosti MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• virus chřipky A účinky léků   enzymologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amidy MeSH
• antivirové látky * MeSH
• endonukleasy * MeSH
• inhibitory enzymů * MeSH

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.

• Klíčová slova
• 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
• antivirové látky * farmakologie   chemie   MeSH
• farmakoterapie COVID-19 MeSH
• HEK293 buňky MeSH
• inhibitory proteas farmakologie   chemie   MeSH
• koronavirové proteasy 3C * metabolismus   antagonisté a inhibitory   chemie   genetika   MeSH
• lidé MeSH
• mutace MeSH
• objevování léků * metody   MeSH
• proteolýza MeSH
• SARS-CoV-2 * enzymologie   účinky léků   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky * MeSH
• inhibitory proteas MeSH
• koronavirové proteasy 3C * MeSH

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml.

• Klíčová slova
• DIANA, FAP quantification, Fibroblast activation protein, High-throughput screening,
• MeSH
• cefalosporiny chemie   farmakologie   MeSH
• endopeptidasy * metabolismus   MeSH
• knihovny malých molekul farmakologie   chemie   MeSH
• lidé MeSH
• membránové proteiny * antagonisté a inhibitory   metabolismus   MeSH
• molekulární struktura MeSH
• rychlé screeningové testy * MeSH
• schvalování léčiv MeSH
• serinové endopeptidasy * metabolismus   MeSH
• Úřad Spojených států pro potraviny a léky MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• želatinasy * antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Spojené státy americké MeSH
• Názvy látek
• cefalosporiny MeSH
• endopeptidasy * MeSH
• fibroblast activation protein alpha MeSH Prohlížeč
• knihovny malých molekul MeSH
• membránové proteiny * MeSH
• serinové endopeptidasy * MeSH
• želatinasy * MeSH

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the transcription factor TCF11/NRF1 (NFE2L1), crucial for maintaining proteostasis in mammalian cells, enabling the expression of rescue factors, including proteasome subunits. Here, we describe the consequences of DDI2 ablation in vivo and in cells. DDI2 knock-out (KO) in mice caused embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of embryos showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates and induction of the unfolded protein response (UPR) and cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activated the integrated stress response (ISR) and induced a type I interferon (IFN) signature and IFN-induced proliferative signaling, possibly ensuring survival. These results indicate an important role for DDI2 in the cell-tissue proteostasis network and in maintaining a balanced immune response.

• Klíčová slova
• Biological sciences, Developmental biology, Immune respons,
• Publikační typ
• časopisecké články 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.

• 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
• Názvy látek
• akrylamidy MeSH
• fluorescenční barviva * MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• polymery MeSH
• proteasy 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.

• Klíčová slova
• HPMA copolymer, PD-1, PD-L1, T-cell, antibody mimetic, immune checkpoint, immunosuppression, immunotherapy, inhibitor, tumor immunology,
• MeSH
• antigeny CD274 * antagonisté a inhibitory   imunologie   metabolismus   MeSH
• inhibitory kontrolních bodů * farmakologie   chemie   MeSH
• lidé MeSH
• monoklonální protilátky chemie   farmakologie   MeSH
• nádorové buněčné linie MeSH
• polymery chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD274 * MeSH
• CD274 protein, human MeSH Prohlížeč
• inhibitory kontrolních bodů * MeSH
• monoklonální protilátky MeSH
• polymery 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

Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.

• Klíčová slova
• FOLH1, N-acetyl-aspartyl-glutamate, folyl-poly-γ-glutamyl hydrolase I, glutamate carboxypeptidase II, lipidomics, metabolomics,
• MeSH
• dipeptidy metabolismus   MeSH
• glutamátkarboxypeptidasa II * genetika   metabolismus   MeSH
• kyselina glutamová MeSH
• lipidomika * MeSH
• lipidy chemie   MeSH
• metabolomika * MeSH
• mozek metabolismus   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dipeptidy MeSH
• Folh1 protein, mouse MeSH Prohlížeč
• glutamátkarboxypeptidasa II * MeSH
• isospaglumic acid MeSH Prohlížeč
• kyselina glutamová MeSH
• lipidy MeSH

Some medically important viruses-including retroviruses, flaviviruses, coronaviruses, and herpesviruses-code for a protease, which is indispensable for viral maturation and pathogenesis. Viral protease inhibitors have become an important class of antiviral drugs. Development of the first-in-class viral protease inhibitor saquinavir, which targets HIV protease, started a new era in the treatment of chronic viral diseases. Combining several drugs that target different steps of the viral life cycle enables use of lower doses of individual drugs (and thereby reduction of potential side effects, which frequently occur during long term therapy) and reduces drug-resistance development. Currently, several HIV and HCV protease inhibitors are routinely used in clinical practice. In addition, a drug including an inhibitor of SARS-CoV-2 main protease, nirmatrelvir (co-administered with a pharmacokinetic booster ritonavir as Paxlovid®), was recently authorized for emergency use. This review summarizes the basic features of the proteases of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and SARS-CoV-2 and discusses the properties of their inhibitors in clinical use, as well as development of compounds in the pipeline.

• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• COVID-19 * MeSH
• HIV infekce * farmakoterapie   MeSH
• lidé MeSH
• SARS-CoV-2 MeSH
• virové proteasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• nirmatrelvir and ritonavir drug combination MeSH Prohlížeč
• virové proteasy MeSH