Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-<b>α</b> converting enzyme as potential therapeutics for hepatitis

<div><p></p><p>The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a <i>N</i>-terminal carboxybenzyl group (chemically protective group) and a <i>C</i>-terminal hydroxamate moiety (coordinated to the Zn²⁺ at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein–peptide affinity predictor and were then tested <i>in vitro</i> with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants <i>K</i>_i of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE–inhibitor complex architecture.</p></div

Effect of gestational zinc deficiency on T cell proliferation.

<p>Splenocytes from each group were obtained on day 14 after the third HBV vaccination and analyzed for the HBsAg-specific T cell proliferative response, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073461#s2" target="_blank">Materials and Methods</a> and expressed as SI. The final concentration in the 96-well plate for ConA, BSA and recombinant HBsAg was 2.5, 30 and 20 µg/ml, respectively. The data are expressed as mean ± S.D. of 3 animals in the proliferative assay from one representative experiment. Each experiment was performed 3 times. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

Effect of gestational zinc deficiency on antibody response.

<p>(A) Gestational zinc deficiency decreases the titers of HBsAg-specific IgG in serum. (B) Decrease of HBsAg-specific IgG1 and IgG2a in zinc deficiency offsprings. (C) Prenatal zinc deficiency lessens the IgG2a/IgG1 ratio. For all experiments, serum samples from BALB/c mice fed with different zinc content diets were collected on day 14 after the third HBV vaccination and the concentration of anti-HBs IgG, IgG1 or IgG2a was determined by ELISA using a standard curve generated from serially diluted control anti-HBs antibodies. The data are expressed as mean ± S.D. of 5 animals from one representative experiment. Each experiment was performed 3 times. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

Immune cell populations in the spleen of offspring mice whose mothers received different assigned diets.

<p>Total splenocytes were counted after removal of red blood cells and stained for T cells (CD3⁺) and B cells (CD19⁺) on day 14 after the third HBV vaccination. Cells were analyzed by flow cytometric analysis. The number of T cells and B cells in spleen of the pups was counted. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p>*<p><i>P</i><0.05 compared with control.</p

Effect of gestational zinc deficiency on HBsAg-specific cytokine expressions in T cell subsets.

<p>Splenocytes isolated from the spleen of offspring BALB/c mice on day 14 after the third HBV vaccination were re-stimulated with recombinant HBsAg. Intracellular staining for IFN-γ, IL-4 in CD4⁺ T cells and IFN-γ in CD8⁺ T cells was performed. The summaries of percentage of IFN-γ in total CD4⁺ (A), IL-4 in total CD4⁺ (B) and IFN-γ in total CD8⁺ T cells (C) are shown (D). The data are expressed as mean ± S.D. of 5 animals in the intracellular cytokine staining assay from one representative experiment. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

Serum zinc concentration (µmol/L) of maternal mice given different assigned diets during pregnancy (A) and their 6-week-old offspring (B).

<p>Serum zinc concentrations measurements were carried out on an Agilent 7700x ICP-MS. The data are expressed as mean ± S.D. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

Ratio of thymus or spleen to body weight in the offspring mice whose mothers received different assigned diets during pregnancy.

<p>The weight of thymus and spleen were recorded after the offspring were sacrificed. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

Effect of gestational zinc deficiency on the secretion of cytokines.

<p>Splenocytes isolated from the spleen of offspring BALB/c mice on day 14 after the third HBV vaccination were stimulated with recombinant HBsAg for 72 h. Culture supernatants were harvested, and the production of IFN-γ (A) and IL-4 (B) was detected by ELISA. The data are expressed as mean ± S.D. for 5 animals in the enumeration of cytokines-secreting from one representative experiment. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p

CD3⁺ T cell population and its CD4⁺ and CD8⁺ T subpopulations.

<p>Total splenocytes were counted after removal of red blood cells and stained for T cell population (CD3⁺), CD4⁺ T cells, and CD8⁺ T cells on day 14 after the third HBV vaccination. Cells were analyzed by flow cytometric analysis. The data above show the numbers and percentages of T cells. ZD: pups from the zinc deficiency group; ZS: pups from the zinc supplemented group.</p>*<p><i>P</i><0.05 compared with control.</p

Differences in clinical and laboratory characteristics between severe and non-severe cases of SFTSV infection.

<p>Data are median (interquartile range).</p>a<p>Severe versus non-severe.</p