PSMA-617 is recognized as a benchmark ligand for prostate-specific membrane antigen (PSMA) owing to its broad utilization in prostate cancer (PCa) targeted radionuclide therapy. In this study, the structure-activity relationships (SAR) of PSMA-617 and two novel analogs featuring modified linkers were investigated. In compounds P17 and P18, the 2-naphthyl-l-Ala moiety was replaced with a less lipophilic 3-styryl-l-Ala moiety while the cyclohexyl ring in P18 was replaced with a phenyl group. The first ever crystal structure of the PSMA/PSMA-617 complex reported here revealed a folded conformation of the PSMA-617 linker while for the PSMA/P17 and PSMA/P18 complexes, the extended orientations of the linkers revealed linker flexibility within the PSMA cavity, a change in binding that can be exploited for the structure-guided design of PSMA-targeting agents. Despite structural differences from PSMA-617, the analogs maintained high PSMA inhibition potency, cellular binding, and internalization. In vivo biodistribution studies revealed comparable tumor uptake across all three compounds with P18 displaying higher spleen accumulation, likely due to phenyl ring lipophilicity. These SAR findings provide a strategic framework for the rational design of PSMA ligands, paving the way for the development of next-generation theranostic agents for PCa.

• Publikační typ
• časopisecké články MeSH

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

• MeSH
• inhibitory proteas * farmakologie   MeSH
• ionty MeSH
• metaloproteiny * chemie   MeSH
• zinek metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteas * MeSH
• ionty MeSH
• metaloproteiny * MeSH
• zinek MeSH

Prostate cancer (PCa) tops the list of cancer-related deaths in men worldwide. Prostate-specific membrane antigen (PSMA) is currently the most prominent PCa biomarker, as its expression levels are robustly enhanced in advanced stages of PCa. As such, PSMA targeting is highly efficient in PCa imaging as well as therapy. For the latter, PSMA-positive tumors can be targeted directly by using small molecules or macromolecules with cytotoxic payloads or indirectly by engaging the immune system of the host. Here we describe the engineering, expression, purification, and biological characterization of bispecific T-cell engagers (BiTEs) that enable targeting PSMA-positive tumor cells by host T lymphocytes. To this end, we designed the 5D3-αCD3 BiTE as a fusion of single-chain fragments of PSMA-specific 5D3 and anti-CD3 antibodies. Detailed characterization of BiTE was performed by a combination of size-exclusion chromatography, differential scanning fluorimetry, and flow cytometry. Expressed in insect cells, BiTE was purified in monodisperse form and retained thermal stability of both functional parts and nanomolar affinity to respective antigens. 5D3-αCD3's efficiency and specificity were further evaluated in vitro using PCa-derived cell lines together with peripheral blood mononuclear cells isolated from human blood. Our data revealed that T-cells engaged via 5D3-αCD3 can efficiently eliminate tumor cells already at an 8 pM BiTE concentration in a highly specific manner. Overall, the data presented here demonstrate that the 5D3-αCD3 BiTE is a candidate molecule of high potential for further development of immunotherapeutic modalities for PCa treatment.

• Publikační typ
• časopisecké články MeSH

Glutamate carboxypeptidase II (GCPII) is a metalloprotease implicated in neurological diseases and prostate oncology. While several classes of potent GCPII-specific inhibitors exist, the development of novel active scaffolds with different pharmacological profiles remains a challenge. Virtual screening followed by in vitro testing is an effective means for the discovery of novel active compounds. Structure- and ligand-based pharmacophore models were created based on a dataset of known GCPII-selective ligands. These models were used in a virtual screening of the SPECS compound library (∼209.000 compounds). Fifty top-scoring virtual hits were further experimentally tested for their ability to inhibit GCPII enzymatic activity in vitro. Six hits were found to have moderate to high inhibitory potency with the best virtual hit, a modified xanthene, inhibiting GCPII with an IC50 value of 353 ± 24 nM. The identification of this novel inhibitory scaffold illustrates the applicability of pharmacophore-based modeling for the discovery of GCPII-specific inhibitors.

• MeSH
• glutamátkarboxypeptidasa II * MeSH
• lidé MeSH
• ligandy MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutamátkarboxypeptidasa II * MeSH
• ligandy MeSH

BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.

• Klíčová slova
• Fasciola hepatica, Folate hydrolase, Immunohistochemistry, M28B metalloproteases, NAALADase, Platyhelminth, Prostate specific-membrane antigen, RNA in situ hybridization, Schistosoma mansoni,
• MeSH
• Fasciola hepatica * genetika   MeSH
• myši MeSH
• proteasy MeSH
• savci MeSH
• Schistosoma mansoni MeSH
• Trematoda * genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glutamate carboxypeptidase MeSH Prohlížeč
• proteasy MeSH

Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.

• Klíčová slova
• ATG4B, biosynthesis, cell death, electron transport chain, endothelial cells, mitochondria, oxidative phosphorylation, oxidative stress, reactive oxygen species,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• autofagie * MeSH
• cystein metabolismus   MeSH
• dextrany metabolismus   MeSH
• dýchání MeSH
• endoteliální buňky metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• formaldehyd metabolismus   MeSH
• fosfatidylethanolaminy metabolismus   MeSH
• idiopatické střevní záněty * metabolismus   MeSH
• isothiokyanatany MeSH
• lidé MeSH
• lipopolysacharidy metabolismus   MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mTORC1 metabolismus   MeSH
• myši MeSH
• proteinkinasy aktivované AMP metabolismus   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• sirolimus MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• cystein MeSH
• dextrany MeSH
• formaldehyd MeSH
• fosfatidylethanolaminy MeSH
• isothiokyanatany MeSH
• lipopolysacharidy MeSH
• mitochondriální DNA MeSH
• mTORC1 MeSH
• phenethyl isothiocyanate MeSH Prohlížeč
• proteinkinasy aktivované AMP MeSH
• proteiny asociované s mikrotubuly MeSH
• reaktivní formy kyslíku MeSH
• sirolimus MeSH

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

• Klíčová slova
• N-glycosylation, NAALADase I, PSMA, folate hydrolase, glutamate carboxypeptidase II, site-specific glycoform,
• MeSH
• antigeny povrchové metabolismus   MeSH
• buněčné linie MeSH
• glutamátkarboxypeptidasa II metabolismus   MeSH
• glykosylace MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové biomarkery analýza   MeSH
• nádory prostaty * metabolismus   patologie   MeSH
• polysacharidy * klasifikace   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• prostata * enzymologie   metabolismus   patologie   MeSH
• staging nádorů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• nádorové biomarkery MeSH
• polysacharidy * MeSH

The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl).Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV.IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.

• Publikační typ
• časopisecké články MeSH

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

• Klíčová slova
• computational saturation, directed evolution, interleukin-10, protein engineering, protein scaffold, ribosome display,
• MeSH
• databáze proteinů MeSH
• interleukin-10 metabolismus   MeSH
• konformace proteinů MeSH
• počítačová simulace MeSH
• proteinové inženýrství MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• ribozomy metabolismus   MeSH
• řízená evoluce molekul metody   MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interleukin-10 MeSH
• proteiny MeSH
• rekombinantní proteiny MeSH

Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein-RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

• MeSH
• antigeny povrchové chemie   MeSH
• aptamery nukleotidové chemie   MeSH
• buňky PC-3 MeSH
• glutamátkarboxypeptidasa II chemie   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• nádorové biomarkery chemie   MeSH
• nádory prostaty metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• A9g RNA aptamer MeSH Prohlížeč
• antigeny povrchové MeSH
• aptamery nukleotidové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• ligandy MeSH
• nádorové biomarkery MeSH