In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy

Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.     

Bayesian Total Internal Reflection Fluorescence Correlation Spectroscopy Reveals hIAPP-Induced Plasma Membrane Domain Organization in Live Cells

Amyloid fibril deposition of human islet amyloid polypeptide (hIAPP) in pancreatic islet cells is implicated in the pathogenesis of type II diabetes. A growing number of studies suggest that small peptide aggregates are cytotoxic via their interaction with the plasma membrane, which leads to membrane permeabilization or disruption. A recent study using imaging total internal reflection-fluorescence correlation spectroscopy (ITIR-FCS) showed that monomeric hIAPP induced the formation of cellular plasma membrane microdomains containing dense lipids, in addition to the modulation of membrane fluidity. However, the spatial organization of microdomains and their temporal evolution were only partially characterized due to limitations in the conventional analysis and interpretation of imaging FCS datasets. Here, we apply a previously developed Bayesian analysis procedure to ITIR-FCS data to resolve hIAPP-induced microdomain spatial organization and temporal dynamics. Our analysis enables the visualization of the temporal evolution of multiple diffusing species in the spatially heterogeneous cell membrane, lending support to the carpet model for the association mode of hIAPP aggregates with the plasma membrane. The presented Bayesian analysis procedure provides an automated and general approach to unbiased model-based interpretation of imaging FCS data, with broad applicability to resolving the heterogeneous spatial-temporal organization of biological membrane systems.     

Benzothiazinones Mediate Killing of Corynebacterineae by Blocking Decaprenyl Phosphate Recycling Involved in Cell Wall Biosynthesis

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.     

Genetic predisposition to left ventricular dysfunction: a multigenic and multi-analytical approach

Background

Left ventricular dysfunction (LVD) is a complex, multifactorial condition, caused by mechanical, neurohormonal, and genetic factors. We have previously observed association of renin–angiotensin–aldosterone system (RAAS), matrix metalloproteinases (MMPs) and inflammatory pathway genes with LVD. Therefore the present study was undertaken to identify the combination of genetic variants and their possible interactions contributing towards genetic susceptibility to LVD in the background of coronary artery disease (CAD).

Methods and results

The study included 230 healthy controls and 510 consecutive patients with angiographically confirmed CAD. Among them, 162 with reduced left ventricle ejection fraction (LVEF ≤ 45%) were categorized as having LVD. We analyzed 11 polymorphisms of RAAS, MMPs and inflammatory pathways. Single locus analysis showed that AT1 A1166C (p value < 0.001; OR = 3.67), MMP9 R668Q (p value = 0.007; OR = 3.48) and NFKB1-94 ATTG ins/del (p value = 0.013; OR = 2.01) polymorphisms were independently associated with LVD when compared with both non-LVD patients and healthy controls. High-order gene–gene interaction analysis, using classification and regression tree (CART) and multifactor dimensionality reduction (MDR) revealed that AT1 A1166C and NFKB1-94 ATTG ins/del polymorphisms jointly increased the risk of LVD to great extent (p-value = 0.001; OR = 8.55) and best four-factor interaction model consisted of AT1 A1166C, MMP7 A-181G, MMP9 R668Q and NFKB1-94 ATTG ins/del polymorphisms with testing accuracy of 0.566 and cross validation consistency (CVC) = 9/10 (permutation p < 0.001) showed increased risk for LVD respectively.

Conclusion

AT1 A1166C independently and in combination with MMP9 R668Q and NFKB1-94 ATTG ins/del polymorphisms plays important role in conferring genetic susceptibility to LVD in CAD patients.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.