GPCR Compound Library

It has become common in the literature for the presence of an activating phosphorylation on a signaling kinase to be interpreted as an ‘active’ kinase pathway. This unfortunate simplification ignores more subtle means of signal regulation. Many signaling events, including UV irradiation, inflammatory cytokines, and mitogen stimulation, are accompanied by generation of reactive oxygen species. These oxidative species include those that acquire one electron from NADH and those that acquire two electrons from NADH. One-electron oxidoreduction reactions result in the release of high-energy species generated by the unused electron from NADH. These species include superoxide, hydroxyl radical, and others, that likely contribute to damaging modifications of proteins and lipids. Twoelectron oxidoreduction mainly results in the in situ generation of hydrogen peroxide, that can be used by endogenous enzymes to oxidize cysteine residues on proteins. These modifications take several forms, including oxidation to sulfenic acid or formation of disulfides involving intra- and intermolecular connections between proteins or S-thiolation of proteins by glutathione and cysteine. Reversible oxidation of proteins on cysteines is potentially a means for controlling signal transduction, since it adds an often-charged, bulky moiety to the protein primary structure, and is reversible, much like protein modifications resulting from phosphorylation. For example, the activity of the MEKK1 protein kinase is regulated by reversible glutathionylation of a single cysteine residue in the ATP binding pocket. Several other kinases have been shown to be activated or inhibited by reversible cysteine oxidation, though mainly with non-physiologic stimuli. Compared to the study of protein phosphorylation, study of specific oxidation on proteins is difficult. Antibodies against Sglutathionylation have been described, and protein sulfhydryls have been labeled with fluorescent maleimides or iodoacetamides. We have developed an unbiased method of quantifying reversible cysteine oxidation in proteins using an affinity capture technique we call Purification of Reversibly Oxidized Proteins. We have applied this technique to study the regulatory oxidation of the p38 MAPK signaling kinase. We have found that p38 is oxidized following exposure of cells to exogenous hydrogen AG-013736 peroxide or prostaglandin J2, and that oxidation results in kinase inactivation despite continued phosphorylation on the activating residues detected by immunoblot. Prostaglandin J2 is an inflammatory mediator that hasbeen characterized forits roleininducing oxidative signals. As such, the oxidation of p38 following exposure of cells to PGJ2 reflects a relevant physiologic signaling event that might occur during inflammation.

We synthesized both T1 and T2 agents to evaluate the ability for each type of probe to visualize inflammation at high resolution in living systems. These probes were utilized for multimodality imaging of vascular plaques in animal models of vascular inflammation to demonstrate the feasibility of using PET screening to guide selection of volumes for high resolution MRI in order to map macrophage distributions in arterial lesions. While no single animal model ideally recreates the human condition, a number of models have shown sufficient parallel to the human disease to justify their use as models for specific disease traits. The difficulty with modeling vulnerable plaques has been that although stenotic lesions can be induced in animal models, they have not been observed to spontaneously rupture as human lesions do. Recent observations of ApoE knockout mice has indicated that in this model, plaques can and do spontaneously rupture. This knockout mouse also has been observed to form lesions similar to the vulnerable human case which are rich in macrophage content. While this model can produce plaques with unstable phenotype, we found it to be inconsistent, producing macrophage-rich plaques in only a fraction of examined animals. Macrophage laden plaques were more reliably produced if a carotid artery was ligated. We used the ApoE ligation model, therefore, as one of the testbeds for our probes. The robustness of the method is demonstrated in that localization of the probe to plaques could be observed in several different animal models of vascular inflammation. We envision that these CT99021 methods could be used as a clinical diagnostic tool wherein PET is used for screening large volumes to identify broad vascular regions that accumulate the macrophage targeted probes. This information is used to guide MR imaging at high resolution to visualize the vessel walls, plaques, and macrophage distribution patterns. Videos are available for this article that demonstrate how the resulting MR data can be presented to provide a type of noninvasive “endoscopy”. Together the information about macrophage density and distribution can be used to estimate plaque probability to rupture and guide patient management decisions. Alone, PET can provide a rough location for macrophage rich plaques and allow some quantitation, but typical plaques are much smaller than the resolution limit of PET, so this method provides only an averaged assessment over all plaques in a given volume, specific information about individual plaque vulnerability is not possible. Human plaques at their largest are a few millimeters thick, up to ten millimeters long and disease is often diffuse throughout vessels. At a resolution of 8–16 mm for whole body scans PET alone is unable to reveal plaque structure but will primarily give general localization. At this resolution it would be difficult to correlate the PET signal with individual stenoses or to determine which regions, specifically, are at risk to rupture. PET is also unable to provide information on exactly degree anywhere in the vascular system.

Intense interest in the pathology of vulnerable plaques has lead to the recognition that plaque composition, more than degree of vessel occlusion, is the primary determinant of stability. Plaques prone to rupture are generally characterized by thin fibrous caps, large, lipid-rich cores, with high macrophage content. Macrophages present in the developing plaque release cytokines and other factors that can weaken the fibrous cap, eventually leading to plaque instability and rupture. In the coronary arteries, numerous reports have observed that high macrophage density is characteristic of lesions vulnerable to rupture. Furthermore, it has been observed that the pattern of distribution of macrophages in the plaque correlates with degree of instability. An ex vivo study of human coronary artery plaque specimens showed that the extent of inflammation at the plaque shoulders appears to correlate with degree of vulnerability slightly unstable plaques have little or no inflammation at the plaque shoulders, while highly unstable plaques have extensive inflammation at the plaque shoulders. Therefore, the ability to image plaques at high resolution to determine macrophage content and distribution could provide a means to noninvasively assess plaque vulnerability and degree of risk to rupture in inflamed arteries. There is currently no clinical method to assess plaque vulnerability in vivo; the ability to do so could provide a critical diagnostic to guide management of patients with vascular disease. The gold standard for imaging atherosclerotic disease is angiography. Angiographic images provide information on decreasing of vessel lumen as plaques invade the luminal space. Highly stenotic plaques may be revealed by this technique; however angiography cannot provide direct assessment of the extent of disease in the vessel wall, nor can it detect disease in vessels that have positive remodeling to enlarge vessel diameter in response to plaque growth. The recognition that the majority of clots leading to acute coronary events occur in plaques that are not highly stenotic highlighted the need for alternative imaging methods that can directly image the vessel wall. There are a number of alternative techniques to image plaques including invasive modalities such as intravascular ultrasound, angioscopy, thermography, optical coherence tomography, raman spectroscopy, near infra-red spectroscopy and intravascular MRI. These invasive techniques involve intravascular transceivers that must be threaded into the vessel being examined and therefore are unsuitable for exploratory imaging to assess overall plaque LDN-193189 burden in the patient. Noninvasive methods are better suited for examining larger regions; ultrasound, computed tomography and magnetic resonance imaging have received the most attention. Ultrasound and computed tomography can provide information about cap thickness and plaque calcification but MRI shows the most promise for assessing both structure and lipid composition to evaluate plaque stability.

Although small differences in T cell proliferation activity and CTL cytotoxicity were observed between hTERTC27 treated mice and control group mice, induced by rAAV-/rAdvhTERTC27 observed in our study. A minor increase of CTL cytotoxicity in spleen lymphocytes might be a result of increased levels of Th1 cytokine in blood. A considerable increase in the levels of IL-2, IFN-c and GM-CSF was observed in the plasma of mice treated with rAAV-/rAdv-hTERTC27 compared with the control mice. All these cytokines function as an immune adjuvant and are known to contribute to the development and activity of tumor specific CTL. However, further investigation is required to determine whether cytokines such as IL-2 and IFN-c could increase the population of cytokine-inducedkiller cells in vivo and contribute to the antitumor effects of hTERTC27. Unlike our previous study in which ectopic expression of rAAV-hTERTC27 in nude mice significantly upregulated the IL-17 mRNA level in xenografted tumor tissue, administration of rAAV-/rAdv-hTERTC27 viral cocktail in C57BL/6 mice showed a little increase of IL-17 cytokine level in blood in this study. The discrepancy may come from the different mouse models and delivery systems used between these two studies. Nonetheless, the slight change in IL-17 level is FG-4592 consistent with the mild increase in activated T cells because it is known that IL-17 expression is restricted to activated T-cells. It is worth noting that NK cells are known to play a major role in cytokine-mediated inhibition of B16 melanoma development and that IL-2 can induce the proliferation and activity of NK cells. Moreover, activated NK cells can secrete several cytokines, including IFN-c and GM-CSF and increase IL-2 mRNA expression. In addition, IFN-c itself also activates NK cells. Because of the complex relationship between NK cells and these cytokines, the initial effect following hTERTC27 administration remains elusive and requires further investigation. Elevated levels accurately reflect the presence of neuropathological conditions including traumatic head injuries, psychiatric disorders, cerebrovascular insults and neurodegenerative diseases, while normal levels reliably exclude major CNS pathology. Its potential clinical use in the therapeutic decision making process is substantiated by a vast body of literature validating variations in serum 100B levels with standard modalities for prognosticating the extent of CNS damage: alterations in neuroimaging, cerebrospinal pressure, and other brain molecular markers. Thus, the major advantage of using S100B is that elevations in serum can be easily measured, providing a sensitive measure to help rule out major CNS dysfunction. An important application of serum S100B testing is the selection of patients with minor head injury who do not need further neuroradiological evaluation, as studies comparing CT scans and S100B levels have demonstrated S100B values below 0.1 ng/mL are associated with low risk of obvious neuroradiological changes or significant clinical sequelae.

The innate arm of immunity mediated by NK cells and adaptive immunity mediated by Th1 cytokines seemed to play an important role in this anti-tumor effect. The potential beneficial effects of supplementing rAAV-/rAdv-hTERTC27 gene therapy with other therapies for prevention and treatment of melanoma metastasis warrant further investigations. nhanced expression level of hTERTC27 likely played a role. As a universal tumor-associated antigen, TERT is an ideal Dinaciclib target for cancer therapy. Conceptually, three approaches have exploited TERT for cancer therapy: gene therapy, immunotherapy, and small-molecule inhibitors. Previously, rAAV-hTERTC27 was developed as a TERT-targeting gene therapy. Our study indicates that activation of NK cells by hTERTC27 is sufficient to inhibit melanoma growth in mouse model, pinpointing the importance of NK cell activation in cancer immunotherapy. These observations are consistent with our previous study that rAAV-hTERTC27 administration effectively inhibited glioblastoma growth in nude mice, where T cells are absent but NK cells are functional. Consistently, a previous study also indicated that activation of NK cells can provide effective innate immunotherapy of melanoma in mouse model. The NK activity-stimulating effect of rAAV-/rAdv-hTERTC27 represents a novel way to exploit the functions of TERT in cancer treatment. hTERTC27 contains two peptides, p973 and p988, both of which have been shown to induce TERT specific CTLs in vitro or in vivo to lyse TERT+ tumor cells, including melanoma B16 cells. It has also been reported that dendritic cells pulsed with peptide p540 can elicit CTLs ex vivo to lyse hTERT+ tumor cells, including human melanoma cells K029. DCs transfected with mTERT gene can also induce CTLs to lyse B16 cells and inhibit B16 melanoma metastasis. Apparently, the mechanism of hTERTC27-induced tumor suppression in our study is different from that of peptides or TERT gene transfected DCs reported in other studies. hTERTC27 may contain other unknown epitopes that have a tendency to induce a strong innate immune response other than an adaptive immune response. Not surprisingly, a single-peptide epitope tends to induce adaptive immunity because such single peptide is screened by CTL assay. However, subcutaneous injection of rAAV-/rAdv-hTERTC27 viral cocktail near tumor site produced the most significant antitumor effect. Although it is unclear what might have contributed to the differences of antitumor effect among these administration routes, it has been reported that the induction of immunity against the transgene product carried by rAAV depends on the routes of administration. Therefore, intratumoral, intravenous or subcutaneous administration of rAAV-/rAdv-hTERTC27 may preferentially enable the stimulation of innate anti-tumor immunity. It will be interesting to investigate the effect of different administration routes on NK cell population and activation. Our observation implies that careful selection of administration route may be important for cancer gene therapy.