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

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

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

HIV protease (PR) is a key target for antiviral drugs, and HIV protease inhibitors (PIs) are a prime example of successful structure-based drug design. PIs show clear therapeutic benefits, but their efficacy can be compromised by poor bioavailabilitity, unwanted side effects, and most importantly, development of antiviral drug resistance. Therefore, the quest for novel, highly active compounds with improved resistance profiles, better pharmacokinetic properties, and fewer adverse effects continues. In particular, the problem of cross-resistance could be circumvented by identifying novel compounds that show different binding modes to HIV PR than the current clinical inhibitors.

• MeSH
• cílená molekulární terapie metody   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemická syntéza   chemie   MeSH
• katalytická doména MeSH
• lidé MeSH
• molekulární struktura MeSH
• peptidomimetika chemická syntéza   chemie   MeSH
• racionální návrh léčiv * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.

• MeSH
• benzodiazepiny chemie   MeSH
• HIV infekce farmakoterapie   enzymologie   virologie   MeSH
• HIV-1 účinky léků   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemická syntéza   farmakologie   MeSH
• katalytická doména MeSH
• katalýza MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• peptidové fragmenty farmakologie   MeSH
• racionální návrh léčiv * MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH