The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

• MeSH
• antigeny nádorové genetika   MeSH
• biotest * MeSH
• DNA účinky léků   genetika   MeSH
• inhibitory karboanhydras izolace a purifikace   terapeutické užití   MeSH
• karboanhydrasa IX antagonisté a inhibitory   genetika   MeSH
• katalytická doména účinky léků   MeSH
• léčivé přípravky MeSH
• lidé MeSH
• rychlé screeningové testy * MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Macrophage cells are present in high abundance in the lung to intercept invading microorganisms that gain access through airway mucosal surfaces. Several bacterial pathogens have evolved the capacity to evade the innate immune response by establishing infections within pulmonary macrophages upon phagocytosis, leading to prolonged disease. Macrolide antibiotics such as azithromycin and clarithromycin accumulate in phagocytic cells and have been shown to preferentially distribute in tissues where populations of these cells reside. We employed this class of molecules as targeting ligands to direct virus-like particles (VLPs) to lung-resident macrophages. VLP-macrolide conjugates showed enhanced uptake into RAW 264.7 macrophage cells in culture, with azithromycin displaying the greatest effect; distinct differences were also observed for different macrocycle structures and orientations on the particle surface. Activation of macrophage cells was stimulated by particle uptake toward an intermediate activation state, in contrast to previous reports using macrolide-functionalized gold nanorods that stimulated a cytotoxic macrophage response. Attached azithromycin was also able to direct VLPs to the lungs in mice, with significant accumulation within 2 h of systemic injection. These results suggest that this new class of bioconjugate could serve as an effective platform for intracellular drug delivery in the context of pulmonary infections.

• MeSH
• Allolevivirus chemie   MeSH
• alveolární makrofágy metabolismus   MeSH
• antibakteriální látky chemie   farmakokinetika   MeSH
• azithromycin chemie   farmakokinetika   MeSH
• cytokiny metabolismus   MeSH
• Escherichia coli genetika   MeSH
• fagocytóza MeSH
• fenotyp MeSH
• klarithromycin chemie   farmakokinetika   MeSH
• myši MeSH
• plíce účinky léků   metabolismus   MeSH
• příprava léků metody   MeSH
• RAW 264.7 buňky MeSH
• systémy cílené aplikace léků metody   MeSH
• tkáňová distribuce MeSH
• virové plášťové proteiny chemie   farmakokinetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Proteases are directly involved in cancer pathogenesis. Expression of fibroblast activation protein (FAP) is upregulated in stromal fibroblasts in more than 90% of epithelial cancers and is associated with tumor progression. FAP expression is minimal or absent in most normal adult tissues, suggesting its promise as a target for the diagnosis or treatment of various cancers. Here, we report preparation of a polymer conjugate (an iBody) containing a FAP-specific inhibitor as the targeting ligand. The iBody inhibits both human and mouse FAP with low nanomolar inhibition constants but does not inhibit close FAP homologues dipeptidyl peptidase IV, dipeptidyl peptidase 9, and prolyl oligopeptidase. We demonstrate the applicability of this iBody for the isolation of FAP from cell lysates and blood serum as well as for its detection by ELISA, Western blot, flow cytometry, and confocal microscopy. Our results show the iBody is a useful tool for FAP targeting in vitro and potentially also for specific anticancer drug delivery.

• MeSH
• ELISA MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   chemie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• polymery chemie   MeSH
• průtoková cytometrie MeSH
• serinové endopeptidasy chemie   MeSH
• western blotting MeSH
• želatinasy antagonisté a inhibitory   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Human diseases are often diagnosed by determining levels of relevant enzymes and treated by enzyme inhibitors. We describe an assay suitable for both ultrasensitive enzyme quantification and quantitative inhibitor screening with unpurified enzymes. In the DNA-linked Inhibitor ANtibody Assay (DIANA), the target enzyme is captured by an immobilized antibody, probed with a small-molecule inhibitor attached to a reporter DNA and detected by quantitative PCR. We validate the approach using the putative cancer markers prostate-specific membrane antigen and carbonic anhydrase IX. We show that DIANA has a linear range of up to six logs and it selectively detects zeptomoles of targets in complex biological samples. DIANA's wide dynamic range permits determination of target enzyme inhibition constants using a single inhibitor concentration. DIANA also enables quantitative screening of small-molecule enzyme inhibitors using microliters of human blood serum containing picograms of target enzyme. DIANA's performance characteristics make it a superior tool for disease detection and drug discovery.

• MeSH
• biotest * MeSH
• DNA * MeSH
• enzymy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• objevování léků * MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

UNLABELLED: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.

• MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• glutamáty chemie   metabolismus   MeSH
• karboxypeptidasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• lidé MeSH
• molekulární struktura MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homologue, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.

• MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   chemie   metabolismus   MeSH
• HEK293 buňky účinky léků   MeSH
• inhibitory enzymů chemie   metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• močovina chemie   MeSH
• molekulární struktura MeSH
• preklinické hodnocení léčiv metody   MeSH
• racionální návrh léčiv MeSH
• techniky syntetické chemie MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

• MeSH
• aminokumariny chemická syntéza   farmakologie   MeSH
• časové faktory MeSH
• fotolýza MeSH
• HEK293 buňky MeSH
• HIV-1 účinky léků   fyziologie   účinky záření   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemická syntéza   farmakologie   MeSH
• karbamáty chemická syntéza   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové prekurzory antagonisté a inhibitory   chemie   metabolismus   MeSH
• proteolýza účinky léků   MeSH
• replikace viru MeSH
• světlo MeSH
• valin analogy a deriváty   chemická syntéza   farmakologie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.

• MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   genetika   metabolismus   MeSH
• inhibitory proteas chemická syntéza   chemie   toxicita   MeSH
• katalytická doména MeSH
• kinetika MeSH
• lidé MeSH
• močovina analogy a deriváty   chemická syntéza   toxicita   MeSH
• nádorové buněčné linie MeSH
• nanočástice chemie   MeSH
• nosiče léků chemie   MeSH
• povrchová plasmonová rezonance MeSH
• racionální návrh léčiv MeSH
• regulace genové exprese účinky léků   MeSH
• rekombinantní proteiny biosyntéza   chemie   genetika   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH