Detection of "Hidden" Antimicrobial Drug Resistance.

Antimicrobial drug resistance has become a serious public health problem. The current clinical diagnostic methods are turbidity-based assays that have been used for years to track bacterial growth; however, the method is relatively insensitive. To eliminate the new occurrence of drug resistance in infectious bacteria, we developed a highly sensitive EZMTT method for the antibiotic susceptibility test (AST) that magnified the cell growth signal and revealed partial drug resistance (showing 2-20% weak cell growth) that was not detected by the current turbidity assay within 24 h. By simply mixing the EZMTT dye with the bacterial culture and then following the growth by absorbance measurement at 450 nm, the drug-induced proliferation (DIP) rate can be obtained in a high-throughput-screening (HTS) mode with greater than 10-fold better sensitivity than the turbidity assay. The EZMTT-based DIP rate assay of 5 clinically isolated E. coli strains found approximately 30% more partial drug resistance than what was detected in the traditional turbidity-based assay. The observed partial drug resistance was further confirmed by mechanistic analyses. Therefore, a combination of the EZMTT dye and the current clinically used VITEK-type technology has great potential to help understand antimicrobial drug resistance and ultimately provide patients with precise medical care to prevent the occurrence of multidrug resistant bacteria.

Kidney-Type Glutaminase Inhibitor Hexylselen Selectively Kills Cancer Cells via a Three-Pronged Mechanism.

Tumor metabolism has been deeply investigated for cancer therapeutics. Here, we demonstrate that glutamine deficiency alone could not completely inhibit cancer cell growth and that many potent kidney-type glutaminase (KGA) inhibitors did not show satisfying in vivo efficacy. The potent KGA allosteric inhibitor, CB-839, resulted in up to 80% growth inhibition of all tested cell lines, whereas Hexylselen (CPD-3B), a KGA/glutamate dehydrogenase (GDH) inhibitor, showed essentially no toxicity to normal cells up to a 10 µM concentration and could completely inhibit the growth of many aggressive cell lines. Further analyses showed that CPD-3B targets not only KGA and GDH but also thioredoxin reductase (TrxR) and amidotransferase (GatCAB), which results in corresponding regulation of Akt/Erk/caspase-9 signaling pathways. In an aggressive liver cancer xenograft model, CPD-3B significantly reduced tumor size, caused massive tumor tissue damage, and prolonged survival rate. These provide important information for furthering the drug design of an effective anticancer KGA allosteric inhibitor.

Cationic Polymethacrylate-Modified Liposomes Significantly Enhanced Doxorubicin Delivery and Antitumor Activity.

Liposome (LP) encapsulation of doxorubicin (DOX) is a clinically validated method for cancer drug delivery, but its cellular uptake is actually lower than the free DOX. Therefore, we modified DOX-LP with a cationic polymer (Eudragit RL100; ER) to improve its cellular uptake and antitumor activity. The resulting DOX-ERLP was a 190 nm nanoparticle that was absorbed efficiently and caused cancer cell death in 5 hrs. Growth as measured by the MTT assay or microscopic imaging demonstrated that DOX-ERLP has at least a two-fold greater potency than the free DOX in inhibiting the growth of a DOX resistant (MCF7/adr) cell and an aggressive liver cancer H22 cell. Further, its in vivo efficacy was tested in H22-bearing mice, where four injections of DOX-ERLP reduced the tumor growth by more than 60% and caused an average of 60% tumor necrosis, which was significantly better than the DOX and DOX-LP treated groups. Our work represents the first use of polymethacrylate derivatives for DOX liposomal delivery, demonstrating the great potential of cationic polymethacrylate modified liposomes for improving cancer drug delivery.

Biomolecular Interaction Assays Identified Dual Inhibitors of Glutaminase and Glutamate Dehydrogenase That Disrupt Mitochondrial Function and Prevent Growth of Cancer Cells.

Glutaminase (KGA/isoenzyme GAC) is an emerging and important drug target for cancer. Traditional methods for assaying glutaminase activity are coupled with several other enzymes. Such coupled assays do not permit the direct and stringent characterization of specific glutaminase inhibitors. Ebselen was identified as a potent 9 nM KGA inhibitor in the KGA/glutamate oxidase (GO)/horse radish peroxidase (HRP) coupled assay but showed very weak activity in inhibiting the growth of glutamine-dependent cancer cells. For rigorous characterization, we developed a direct kinetic binding assay for KGA using bio-layer interferometry (BLI) as the detection method; Ebselen was identified as a GDH inhibitor but not a KGA inhibitor. Furthermore, we designed and synthesized several benzo[d][1,2]selenazol-3(2H)-one dimers which were subjected to SAR analysis by several glutaminolysis specific biochemical and cell based assays. Novel glutamate dehydrogenase (GDH) or dual KGA/GDH inhibitors were discovered from the synthetic compounds; the dual inhibitors completely disrupt mitochondrial function and demonstrate potent anticancer activity with a minimum level of toxicity.

Mono-sulfonated tetrazolium salt based NAD(P)H detection reagents suitable for dehydrogenase and real-time cell viability assays.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of L-glutamate and is important for several biological processes. For GDH inhibitor screening, we developed a novel mono-sulfonated tetrazolium salt (EZMTT), which can be synthesized using H2O2 oxidation and purified easily on silica gel in large quantities. The EZMTT detection method showed linear dose responses to NAD(P)H, dehydrogenase concentration and cell numbers. In E. coli GDH assay, the EZMTT method showed excellent assay reproducibility with a Z factor of 0.9 and caused no false positives in the presence of antioxidants (such as BME). Using the EZMTT-formazan-NAD(P)H system, we showed that EGCG is a potent E. coli GDH inhibitor (IC50 45 nM) and identified that Ebselen, a multifunctional thioredoxin reductase inhibitor, inactivated E. coli GDH (IC50 213 nM). In cell-based assays at 0.5 mM tetrazolium concentration, EZMTT showed essentially no toxicity after a 3-day incubation, whereas 40% of inhibition was observed for WST-8. In conclusion, EZMTT is a novel tetrazolium salt which provides improved features that are suitable for dehydrogenases and real-time cell-based high-throughput screening (HTS).