Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

In silico and in vitro methods to identify ebola virus VP35-dsRNA inhibitors.

Ebola virus continues to be problematic as sporadic outbreaks in Africa continue to arise, and as terrorist organizations have considered the virus for bioterrorism use. Several proteins within the virus have been targeted for antiviral chemotherapy, including VP35, a dsRNA binding protein that promotes viral replication, protects dsRNA from degradation, and prevents detection of the viral genome by immune complexes. To augment the scope of our antiviral research, we have now employed molecular modeling techniques to enrich the population of compounds for further testing in vitro. In the initial docking of a static VP35 structure with an 80,000 compound library, 40 compounds were selected, of which four compounds inhibited VP35 with IC50 <200µM, with the best compounds having an IC50 of 20µM. By superimposing 26 VP35 structures, we determined four aspartic acid residues were highly flexible and the docking was repeated under flexible parameters. Of 14 compounds chosen for testing, five compounds inhibited VP35 with IC50 <200µM and one compound with an IC50 of 4µM. These studies demonstrate the value of docking in silico for enriching compounds for testing in vitro, and specifically using multiple structures as a guide for detecting flexibility and provide a foundation for further development of small molecule inhibitors directed towards VP35.

Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.

Recessively inherited loss-of-function mutations in the PTEN-induced putative kinase 1(Pink1), DJ-1 (Park7) and Parkin (Park2) genes are linked to familial cases of early-onset Parkinson's disease (PD). As part of its strategy to provide more tools for the research community, The Michael J. Fox Foundation for Parkinson's Research (MJFF) funded the generation of novel rat models with targeted disruption ofPink1, DJ-1 or Parkin genes and determined if the loss of these proteins would result in a progressive PD-like phenotype. Pathological, neurochemical and behavioral outcome measures were collected at 4, 6 and 8months of age in homozygous KO rats and compared to wild-type (WT) rats. Both Pink1 and DJ-1 KO rats showed progressive nigral neurodegeneration with about 50% dopaminergic cell loss observed at 8 months of age. ThePink1 KO and DJ-1 KO rats also showed a two to three fold increase in striatal dopamine and serotonin content at 8 months of age. Both Pink1 KO and DJ-1 KO rats exhibited significant motor deficits starting at 4months of age. However, Parkin KO rats displayed normal behaviors with no neurochemical or pathological changes. These results demonstrate that inactivation of the Pink1 or DJ-1 genes in the rat produces progressive neurodegeneration and early behavioral deficits, suggesting that these recessive genes may be essential for the survival of dopaminergic neurons in the substantia nigra (SN). These MJFF-generated novel rat models will assist the research community to elucidate the mechanisms by which these recessive genes produce PD pathology and potentially aid in therapeutic development.

Leptin dependent changes in the expression of tropomyosin receptor kinase B protein in nucleus of the solitary tract to acute intermittent hypoxia.

To investigate the possibility that leptin exerts an effect in NTS by inducing changes in the expression of pre- and/or post-synaptic proteins, experiments were done in Sprague-Dawley wild-type rats (WT) rats and leptin-deficient rats (Lep(Δ151/Δ151); KILO rat) exposed to 8h of continuous intermittent hypoxia (IH) or normoxia. Protein was extracted from the caudal medial NTS and analyzed by western blot for the expression of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), synaptophysin, synaptopodin and growth-associated protein-43 (GAP-43). In WT rats, BDNF and GAP 43 protein expression levels were not altered after IH or normoxia, although there was a trend towards an increase in BDNF expression. On the other hand, after IH, protein expression of both isoforms of the BDNF receptor TrkB (gp95 and gp145) was higher. Furthermore, synaptophysin protein expression was lower compared to normoxic WT rats. In the KILO rat, no changes were observed in the protein expression of BDNF, TrkB, or GAP 43 after IH when compared to KILO normoxic controls. However, synaptophysin was lower in the IH exposed KILO rat compared to normoxic controls, as found in the WT rat. Expression of synaptopodin was not detected in NTS in either IH or normoxic animals of all groups. These results suggest that leptin released during IH may contribute to neurotrophic changes occurring within NTS and that these changes may be associated with altered chemoreceptor reflex function.

Efficient catalytic cycloalkane oxidation employing a "helmet" phthalocyaninato iron(III) complex.

We have examined the catalytic activity of an iron(III) complex bearing the 14,28-[1,3-diiminoisoindolinato]phthalocyaninato (diiPc) ligand in oxidation reactions with three substrates (cyclohexane, cyclooctane, and indan). This modified metallophthalocyaninato complex serves as an efficient and selective catalyst for the oxidation of cyclohexane and cyclooctane, and to a far lesser extent indan. In the oxidations of cyclohexane and cyclooctane, in which hydrogen peroxide is employed as the oxidant under inert atmosphere, we have observed turnover numbers of 100.9 and 122.2 for cyclohexanol and cyclooctanol, respectively. The catalyst shows strong selectivity for alcohol (vs. ketone) formation, with alcohol to ketone (A/K) ratios of 6.7 and 21.0 for the cyclohexane and cyclooctane oxidations, respectively. Overall yields (alcohol + ketone) were 73% for cyclohexane and 92% for cyclooctane, based upon the total hydrogen peroxide added. In the catalytic oxidation of indan under similar conditions, the TON for 1-indanol was 10.1, with a yield of 12% based upon hydrogen peroxide. No 1-indanone was observed in the product mixture.

Resolution of enantiomers of a series of chiral alkoxy-modified phthalocyaninato nickel(II) complexes by enantioselective HPLC.

We have resolved the enantiomers of a series of chiral modified metallophthalocyaninato complexes of nickel bearing alkoxy groups at the 14 and 28 positions on what would otherwise be a normal phthalocyaninato ligand and conforming to the general formula [14,28-(RO)(2)Pc]Ni(ii), where R = Me, Et, or n-Pr. The complex for which R = n-Pr is reported here for the first time. Resolution of the enantiomers of these complexes was accomplished via HPLC utilizing an immobilized carbohydrate-based stationary phase, resulting in baseline resolution of peaks corresponding to enantiomers of the complexes, with R(s) values in excess of five. Isolation of milligram quantities of the complexes bearing methoxy and n-propoxy groups in high enantiomeric excess has been achieved via semi-preparative-scale HPLC on the same stationary phase. Resolved samples of these compounds do not appear to racemize at an appreciable rate, nor do they readily exchange alkoxy groups with alcohols while stirring in alcoholic solution. The spectroscopic details and the crystallographically-determined solid-state structure for the complex where R = n-Pr are reported, and are highly similar to those that have been observed for the previously reported analogues. It has been shown by NMR that the chirality and C(2) molecular symmetry of the complex bearing n-propoxy groups is maintained in solution.