A Mutational Analysis of Residues Essential for Ligand Recognition at the Human P2Y 1 Receptor

We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1 receptor. Mutated receptors were expressed in COS-7 cells with an epitope tag that permitted confirmation of expression in the plasma membrane, and agonist-promoted inositol phosphate accumulation was assessed as a measure of receptor activity. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to be involved in ligand recognition were replaced with alanine and, in some cases, by other amino acids. The potent P2Y1 receptor agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing the R128A, R310A, and S314A mutant receptors, and a markedly reduced potency of 2-MeSATP was observed with the K280A and Q307A mutants. These results suggest that residues on the exofacial side of TM3 and TM7 are critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or maximal effect of 2-MeSATP with the S317A mutant receptor. Alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7–18-fold reduction in potency compared with that observed with the wild-type receptor. These residues thus seem to subserve a less important modulatory role in ligand binding to the P2Y1 receptor. Because changes in the potency of 2-methylthio-ADP and 2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at these mutant receptors, the β- and γ-phosphates of the adenine nucleotides seem to be less important than the α-phosphate in ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in diphosphate or monophosphate potency. This result may be indicative of a greater role of these TM5 residues in γ-phosphate recognition. Taken together, the results suggest that the adenosine and α-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor

Concentration-Dependent Modulation of Amyloid-␤ in Vivo and in Vitro Using the ␥-Secretase Inhibitor, LY-450139

ABSTRACT LY-450139 is a ␥-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-␤ (A␤) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A␤ responses to LY-450139 in the guinea pig, a nontransgenic model that has an A␤ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma A␤ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A␤ levels at early time points, with return to baseline within hours. Higher doses inhibited A␤ levels in all compartments at early time points, but elevated plasma A␤ levels at later time points. To determine whether this phenomenon occurs under steadystate drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma A␤ was significantly inhibited at 10 -30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A␤ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A␤ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the ␥-secretase complex, eliciting A␤ lowering at high concentrations but A␤ elevation at low concentrations. The pathological accumulation of amyloid-␤ peptide into dense core plaques in the brains of Alzheimer's disease patients is the ultimate target of multiple disease-modifying drug discovery efforts. One strategy that has entered the clinic is the use of a ␥-secretase inhibitor to reduce central A␤ production. Preclinically, multiple ␥-secretase inhibitors have demonstrated central and peripheral A␤-lowering activity in transgenic mouse lines overexpressing human mutant amyloid precursor protein The ability of plasma and CSF A␤ to track pharmacological changes in brain A␤ provides a useful method for tracking the efficacy of ␥-secretase inhibitors in the clinic. Because each compartment may have varying degrees of drug exposure and different clearance rates for both drug and A␤, it is important to understand the dynamics of A␤ in each compartment. Dose-response and time course studies with ␥-secretase inhibitors in transgenic mice have revealed difArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.110700. ABBREVIATIONS

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

An examination of deoxyadenosine 5′(α-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y(1) receptor

1. The functional activity of deoxyadenosine 5′(α-thio)triphosphate (dATPαS) was assessed at the cloned human P2Y(1) receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. Cells expressing the receptor responded to adenine nucleotides with an increase in [(3)H]-inositol phosphate accumulation. Half-maximal responses were obtained at approximately 30 nM for 2-methylthioadenosine-5′-triphosphate (2MeSATP), 300 nM for dATPαS, and 1000 nM for adenosine 5′-triphosphate (ATP). dATPαS produced a maximal response that was only 37±4% of that produced by ATP or 2MeSATP. dATPαS also competitively antagonized the phospholipase C response to 2MeSATP with a K(B) of 644±14 nM. Thus dATPαS acts as a low potency partial agonist at P2Y(1) receptors. 3. The selectivity of dATPαS for P2Y(1) receptors was determined by examining its capacity to activate P2Y(2), P2Y(4) and P2Y(6) receptors also stably expressed in 1321N1 cells. Although dATPαS was a partial agonist at P2Y(1) receptors it was a full agonist at P2Y(2) receptors, albeit with a potency that was two orders of magnitude lower than at P2Y(1) receptors. No agonist or antagonist activity was observed at P2Y(4) and P2Y(6) receptors. 4. Although [(35)S]-dATPαS bound to a relatively high density (ca 10 pmol mg(−1) protein) of binding sites in membranes from 1321N1 or Cos-7 cells expressing the P2Y(1) receptor, no difference in the total density of sites was observed between membranes from wild-type, empty vector-transfected, or P2Y(1) receptor-expressing cells. Moreover, adenine nucleotide analogues inhibited [(35)S]-dATPαS binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y(1) receptor-expressing cells. Saturation binding experiments demonstrated multiple affinity states for [(35)S]-dATPαS binding in wild-type Cos-7 cell membranes. These data from 1321N1 and Cos-7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [(35)S]-dATPαS that are not related to P2Y receptor subtypes

Fidelity in functional coupling of the rat P2Y(1) receptor to phospholipase C

1. The rat homologue of the P2Y(1) receptor has been heterologously expressed in 1321N1 human astrocytoma cells and in C6 rat glioma cells. 2. As has been shown previously for the turkey and human P2Y(1) receptors, the rat P2Y(1) receptor expressed in either cell type responded to 2MeSATP with increases in inositol phosphate accumulation that were competitively blocked by the antagonist PPADS. Neither of the wild type cell lines exhibited inositol phosphate responses to P2Y(1) receptor agonists. 3. Expression of the rat P2Y(1) receptor did not confer a capacity of 2MeSATP to inhibit adenylyl cyclase activity in 1321N1 cells. Moreover, the inhibition of adenylyl cyclase mediated by an endogenous P2Y receptor of C6 glioma cells was not enhanced by expression of the rat P2Y(1) receptor. The P2Y receptor-mediated inhibition of adenylyl cyclase in C6 glioma cells expressing both the endogenous P2Y receptor and the rat P2Y(1) receptor remained unaffected by PPADS. 4. Since the P2Y receptor responsible for inhibition of adenylyl cyclase in C6 glioma cells does not share the pharmacological or functional properties of the P2Y(1) receptor, even when both receptors originate from the same species and are simultaneously expressed in the same cell line, it is concluded that the P2Y(1) receptor is distinct from an endogenous P2Y receptor in C6 cells that couples to inhibition of adenylyl cyclase

Early intervention of tau pathology prevents behavioral changes in the rTg4510 mouse model of tauopathy

<div><p>Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.</p></div