4-Amino-2-Sulfanylbenzoic Acid as a Potent Subclass B3 Metallo-ß-Lactamase-Specific Inhibitor Applicable for Distinguishing Metallo-ß-Lactamase Subclasses.

The number of cases of infection with carbapenem-resistant Enterobacteriaceae (CRE) has been increasing and has become a major clinical and public health concern. Production of metallo-ß-lactamases (MBLs) is one of the principal carbapenem resistance mechanisms in CRE. Therefore, developing MBL inhibitors is a promising strategy to overcome the problems of carbapenem resistance conferred by MBLs. To date, the development and evaluation of MBL inhibitors have focused on subclass B1 MBLs but not on B3 MBLs. In the present study, we searched for B3 MBL (specifically, SMB-1) inhibitors and found thiosalicylic acid (TSA) to be a potent inhibitor of B3 SMB-1 MBL (50% inhibitory concentration [IC50], 0.95 µM). TSA inhibited the purified SMB-1 to a considerable degree but was not active against Escherichia coli cells producing SMB-1, as the meropenem (MEM) MIC for the SMB-1 producer was only slightly reduced with TSA. We then introduced a primary amine to TSA and synthesized 4-amino-2-sulfanylbenzoic acid (ASB), which substantially reduced the MEM MICs for SMB-1 producers. X-ray crystallographic analyses revealed that ASB binds to the two zinc ions, Ser221, and Thr223 at the active site of SMB-1. These are ubiquitously conserved residues across clinically relevant B3 MBLs. ASB also significantly inhibited other B3 MBLs, including AIM-1, LMB-1, and L1. Therefore, the characterization of ASB provides a starting point for the development of optimum B3 MBL inhibitors.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion.

BACKGROUND: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. METHODS: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively. RESULTS: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. CONCLUSIONS: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.