Rhodanine as a Potent Scaffold for the Development of Broad-Spectrum Metallo-β-lactamase Inhibitors

A series of rhodanines was constructed, their Z-configuration was confirmed by small molecule X-ray crystal structures, and their activity against metallo-β-lactamases (MβLs) was measured. The obtained 26 molecules and a thioenolate specifically inhibited the MβL L1 with an IC₅₀ range of 0.02–1.7 μM, and compounds <b>2h</b>–<b>m</b> exhibited broad-spectrum inhibition of the MβLs NDM-1, VIM-2, ImiS, and L1 with IC₅₀ values <16 μM. All inhibitors increased the antimicrobial effect of cefazolin against <i>E. coli</i> cells expressing L1, resulting in a 2–8-fold reduction in MIC. Docking studies suggested that the nitro (NDM-1, CphA, and L1) or carboxyl group (VIM-2) of <b>2l</b> coordinates one or two Zn­(II) ions, while the <i>N</i>-phenyl group of the inhibitor enhances its hydrophobic interaction with MβLs. These studies demonstrate that the diaryl-substituted rhodanines are good scaffolds for the design of future broad-spectrum inhibitors of MβLs

Immunohistochemistric stains were used to identify the location of the putative proteins.

<p>The up-regulated proteins, GRP78 and GSTpi, were specifically located on the gastric adenocarcinoma cells. Contrarily, the down-regulated proteins including ApoAI and A1AT were present on the normal gastric glands. Another down-regulated protein, GKN-1, was shown to appear on the mucosa of gastric tissue.</p

The correlation among the selected proteins.

<p>The GRP78 in these 12 pairs of tissues was expressed correlatively with that of ApoAI (r2: −0.61) and A1AT (r2: −0.49) individually. Meanwhile, ApoAI was expressed correlatively with that of A1AT (r2: 0.62). However, GSTpi and GKN-1 seemed to be independent. **p<0.01. *p<0.05.</p

The correlations between clinical stages of gastric cancer and the expressions of proteins.

<p>Although GRP78 and GSTpi were increased in different stages of gastric cancer, no statistical significance could be found. A trend of increase with clinical stage was found in GRP78.</p

Carbamylmethyl Mercaptoacetate Thioether: A Novel Scaffold for the Development of L1 Metallo-β-lactamase Inhibitors

Given the clinical importance of metallo-β-lactamases (MβLs), a new scaffold, N-substituted carbamylmethyl mercaptoacetate thioether, was constructed. The obtained molecules <b>1</b>–<b>16</b> inhibited MβLs from all three subclasses, but preferentially L1 from subclass B3. Compound <b>9</b> with a <i>p</i>-carboxyphenyl substituent exhibited the broadest spectrum with at least 70% inhibition of enzymes from all subclasses at 100 μM, while compound <b>5</b> with a <i>p</i>-methylphenyl substituent was the most potent inhibitor of any individual enzyme, with 97% inhibition at 100 μM and an IC₅₀ value of 0.41 μM against L1. Isothermal titration calorimetry assays corroborate findings from UV–vis spectrophotometric assays that the inhibition of L1 by <b>5</b> is dose-dependent. Docking studies suggest that the carboxyl group, the sulfide atom, and the carbonyl group of the carbamyl coordinate Zn2 in a chelating fashion. Using <i>E. coli</i> cells expressing L1, <b>6</b> and <b>8</b> were able to decrease cefazolin minimum inhibitory concentration 8-fold

The 2D-DIGE results paired tissue samples.

<p>The differential proteins presented on gels. (left): normal; (right): cancer. Thirty-six proteins were picked by DeCyder statistical software, but only fifteen proteins were identified by PMF or PFF technique. The gel conditions: pI: 4–7; gel: 4–12%.The analytical ranges were from pI 4 to 7 forisoelectric focusing and from 4% to 12% gradient gel for sodium dodecyl sulfate- polyacrylamide gelelectrophoresis.</p

The detailed comparison between normal and tumorous tissue from 2D-DIGE.

<p>The selected putative proteins were presented as stereopictures and enlarged scales gel images. These proteins were selected according to the significant difference below 0.05 (<i>p</i><0.05). In addition, the duplicated identification of ATPB, PDIRP5, ApoAI and GSTpi on adjacent spots indicated that they had a different modification.</p

Discovery of Tumor Markers for Gastric Cancer by Proteomics - Figure 3

<p>(A) By Western blotting, 12 paired of gastric cancer and normal tissues was used to validate the putative proteins including GRP78, GSTpi, A1AT, ApoAI and GKN-1. (B) The GSTpi and GRP78 were significantly over-expressed in gastric cancer tissues.Down-expressions of A1AT, ApoAI and GKN-1 were noted. **<i>p</i><0.01. *<i>p</i><0.05.</p