Structure-based discovery of novel P-glycoprotein inhibitors targeting the nucleotide binding domains.

P-glycoprotein (P-gp), a membrane transport protein overexpressed in certain drug-resistant cancer cells, has been the target of numerous drug discovery projects aimed at overcoming drug resistance in cancer. Most characterized P-gp inhibitors bind at the large hydrophobic drug binding domain (DBD), but none have yet attained regulatory approval. In this study, we explored the potential of designing inhibitors that target the nucleotide binding domains (NBDs), by computationally screening a large library of 2.6 billion synthesizable molecules, using a combination of machine learning-guided molecular docking and molecular dynamics (MD). 14 of the computationally best-scoring molecules were subsequently tested for their ability to inhibit P-gp mediated calcein-AM efflux. In total, five diverse compounds exhibited inhibitory effects in the calcein-AM assay without displaying toxicity. The activity of these compounds was confirmed by their ability to decrease the verapamil-stimulated ATPase activity of P-gp in a subsequent assay. The discovery of these five novel P-gp inhibitors demonstrates the potential of in-silico screening in drug discovery and provides a new stepping point towards future potent P-gp inhibitors.

Phloretin inhibits glucose transport and reduces inflammation in human retinal pigment epithelial cells.

During age-related macular degeneration (AMD), chronic inflammatory processes, possibly fueled by high glucose levels, cause a breakdown of the retinal pigment epithelium (RPE), leading to vision loss. Phloretin, a natural dihydroxychalcone found in apples, targets several anti-inflammatory signaling pathways and effectively inhibits transporter-mediated glucose uptake. It could potentially prevent inflammation and cell death of RPE cells through either direct regulation of inflammatory signaling pathways or through amelioration of high glucose levels. To test this hypothesis, ARPE-19 cells were incubated with or without phloretin for 1 h before exposure to lipopolysaccharide (LPS). Cell viability and the release of pro-inflammatory cytokines interleukin 6 (IL-6), IL-8 and vascular endothelial growth factor (VEGF) were measured. Glucose uptake was studied using isotope uptake studies. The nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were determined alongside the phosphorylation levels of mitogen-activated protein kinases. Phloretin pretreatment reduced the LPS-induced release of IL-6 and IL-8 as well as VEGF. Phloretin increased intracellular levels of reactive oxygen species and nuclear translocation of Nrf2. It also inhibited glucose uptake into ARPE-19 cells and the phosphorylation of Jun-activated kinase (JNK). Subsequent studies revealed that Nrf2, but not the inhibition of glucose uptake or JNK phosphorylation, was the main pathway of phloretin's anti-inflammatory activities. Phloretin was robustly anti-inflammatory in RPE cells and reduced IL-8 secretion via activation of Nrf2 but the evaluation of its potential in the treatment or prevention of AMD requires further studies.

SNAT2 is responsible for hyperosmotic induced sarcosine and glycine uptake in human prostate PC-3 cells.

Solute carriers (SLC) are important membrane transport proteins in normal and pathophysiological cells. The aim was to identify amino acid SLC(s) responsible for uptake of sarcosine and glycine in prostate cancer cells and investigate the impact hereon of hyperosmotic stress. Uptake of 14C-sarcosine and 3H-glycine was measured in human prostate cancer (PC-3) cells cultured under isosmotic (300 mOsm/kg) and hyperosmotic (500 mOsm/kg) conditions for 24 h. Hyperosmotic culture medium was obtained by supplementing the medium with 200 mM of the trisaccharide raffinose. Amino acid SLC expression was studied using RT-PCR, real-time PCR, and western blotting. siRNA knockdown of SNAT2 was performed. Experiments were conducted in at least 3 independent cell passages. The uptake of Sar and Gly was increased approximately 8-ninefold in PC-3 cells after 24 h hyperosmotic culture. PAT1 mRNA and protein could not be detected, while SNAT2 was upregulated at the mRNA and protein level. Transfection with SNAT2-specific siRNA reduced Vmax of Sar uptake from 2653 ± 38 to 513 ± 38 nmol mg protein-1 min-1, without altering the Km value (3.19 ± 0.13 vs. 3.42 ± 0.71 mM), indicating that SNAT2 is responsible for at least 80% of Sar uptake in hyperosmotic cultured PC-3 cells. SNAT2 is upregulated in hyperosmotic stressed prostate cancer cells and SNAT2 is responsible for cellular sarcosine and glycine uptake in hyperosmotic cultured PC-3 cells. Sar is identified as a substrate for SNAT2, and this has physiological implications for understanding cellular solute transport in prostate cancer cells.