Design of Highly Potent Urea-Based, Exosite-Binding Inhibitors Selective for Glutamate Carboxypeptidase II

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

Stimulated Emission Depletion Nanoscopy Reveals Time-Course of Human Immunodeficiency Virus Proteolytic Maturation

Concomitant with human immunodeficiency virus type 1 (HIV-1) budding from a host cell, cleavage of the structural Gag polyproteins by the viral protease (PR) triggers complete remodeling of virion architecture. This maturation process is essential for virus infectivity. Electron tomography provided structures of immature and mature HIV-1 with a diameter of 120–140 nm, but information about the sequence and dynamics of structural rearrangements is lacking. Here, we employed super-resolution STED (stimulated emission depletion) fluorescence nanoscopy of HIV-1 carrying labeled Gag to visualize the virion architecture. The incomplete Gag lattice of immature virions was clearly distinguishable from the condensed distribution of mature protein subunits. Synchronized activation of PR within purified particles by photocleavage of a caged PR inhibitor enabled time-resolved <i>in situ</i> observation of the induction of proteolysis and maturation by super-resolution microscopy. This study shows the rearrangement of subviral structures in a super-resolution light microscope over time, outwitting phototoxicity and fluorophore bleaching through synchronization of a biological process by an optical switch

Concentration dependence of the inflex point of the CSM curves.

<p>The data consist of two independent experimental series (blue and red circles). Linear-regression line of the slope of 76.3 MPa and intercept of 45 MPa is shown which is used to determine the constants <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.92 <i>μM</i> and Δ<i>V</i>_<i>r</i> = 32.5 <i>ml mol</i>⁻¹. Regression lines of the individual series are not shown as they are almost identical; their slopes and intercepts are 77.3 MPa and 43 MPa, respectively (blue), and 74.7 MPa and 47 MPa, respectively (red), which corresponds with <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.95 <i>μM</i>, Δ<i>V</i>_<i>r</i> = 32.1 <i>ml mol</i>⁻¹ (red) and <i>K</i>_{<i>d</i>,<i>atm</i>} = 0.88 <i>μM</i>, Δ<i>V</i>_<i>r</i> = 33.2 <i>ml mol</i>⁻¹ (blue). The error bars of the individual measurements are calculated from the errors of the regression parameters of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.e028" target="_blank">Eq. 19</a> using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.e029" target="_blank">Eq. 20</a> and the error-transition law. [<i>M</i>₀] is considered to be a dimensionless quantity related to the units of μM ([<i>M</i>₀] → [<i>M</i>₀]/<i>μM</i>).</p

Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid Composition

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

Reversibility assay of HIV-1 PR of 5 μM concentration (expressed as dimer).

<p>For every enzyme sample three CSM values were determined: at 10 MPa before pressurizing, at the selected target pressure from the interval of 100 to 350 MPa after 50 min incubation and again at 10 MPa after releasing the pressure. The experiment indicates good reversibility of CSM up to 300 MPa, above this value the reversibility is perturbed by protein aggregation.</p

Model of the HIV-1 PR backbone in front (left) and side (right) view (structure taken from RSCB protein data bank, PDB ID: 1OHR).

<p>The residues of fluorescent amino acids, Trp6, Trp42 and Tyr59, are indicated. Trp6 is located close to the dimerization interface, therefore its spectroscopic properties are probably influenced by dimer dissociation. On the contrary, Trp42 and Tyr59 are located at the most distant site from the dimerization interface, therefore their fluorescence is probably unaffected by dimer dissociation, provided that the monomer keeps its conformation. Trp42 and Tyr59 can eventually partially influence this effect due to the change of their mutual configuration because the conformation of this part of the molecule undergoes partial changes with growing pressure, as was shown by the molecular-dynamics simulation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119099#pone.0119099.ref032" target="_blank">32</a>]. All the fluorophores can contribute to the spectral changes induced by unfolding of monomers or protein aggregation when they undergo more extensive changes of their environment.</p

Parallel Metabolomics and Lipidomics of a PSMA/GCPII Deficient Mouse Model Reveal Alteration of NAAG Levels and Brain Lipid Composition

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

Time development of the tryptophan-fluorescence CSM.

<p>A. Time series for different pressures, each started with a new enzyme sample, concentration of 10 μM dimer. The individual curves are shifted up or down by artificially chosen constant for the sake of better orientation in the graph. B. Time series measured with the same sample of 2 μM concentration, pressure setting is facilitated by pressure jumps. The CSM scale is genuine for all the series.</p

Fluorescence-intensity indicated transition for 5 μM dimer.

<p>A. Time dependence of the intensity for different pressures. Each series is plotted together with the fitting single-exponential decay curve. The same sample was used for the whole set of measurements, pressure setting was facilitated by pressure jumps. The numbers indicate pressure in MPa. B. Equilibrium values of fluorescence for the same experimental series determined from the limit of the fitting curves for time tending to infinity. Inflex point is indicated by the open triangle.</p

Equilibrium values of CSM of ANS emission spectrum for 10 μM dimer fitted by a model function.

<p>Values for the reverse course of pressure indicate irreversibility of the transition.</p