Cytosolic phospholipaseA2 inhibition with PLA-695 radiosensitizes tumors in lung cancer animal models.

Lung cancer remains the leading cause of cancer deaths in the United States and the rest of the world. The advent of molecularly directed therapies holds promise for improvement in therapeutic efficacy. Cytosolic phospholipase A2 (cPLA2) is associated with tumor progression and radioresistance in mouse tumor models. Utilizing the cPLA2 specific inhibitor PLA-695, we determined if cPLA2 inhibition radiosensitizes non small cell lung cancer (NSCLC) cells and tumors. Treatment with PLA-695 attenuated radiation induced increases of phospho-ERK and phospho-Akt in endothelial cells. NSCLC cells (LLC and A549) co-cultured with endothelial cells (bEND3 and HUVEC) and pre-treated with PLA-695 showed radiosensitization. PLA-695 in combination with irradiation (IR) significantly reduced migration and proliferation in endothelial cells (HUVEC & bEND3) and induced cell death and attenuated invasion by tumor cells (LLC &A549). In a heterotopic tumor model, the combination of PLA-695 and radiation delayed growth in both LLC and A549 tumors. LLC and A549 tumors treated with a combination of PLA-695 and radiation displayed reduced tumor vasculature. In a dorsal skin fold model of LLC tumors, inhibition of cPLA2 in combination with radiation led to enhanced destruction of tumor blood vessels. The anti-angiogenic effects of PLA-695 and its enhancement of the efficacy of radiotherapy in mouse models of NSCLC suggest that clinical trials for its capacity to improve radiotherapy outcomes are warranted.

In vitro and in vivo evaluation of a 64Cu-labeled NOTA-Bn-SCN-Aoc-bombesin analogue in gastrin-releasing peptide receptor expressing prostate cancer.

INTRODUCTION: Bombesin (BN) is an amphibian peptide that binds to the gastrin-releasing peptide receptor (GRPR). It has been demonstrated that BN analogues can be radiolabeled for potential diagnosis and treatment of GRPR-expressing malignancies. Previous studies have conjugated various chelators to the eight C-terminal amino acids of BN [BN(7-14)] for radiolabeling with 64Cu. Recently, (1,4,7-triazacyclononane-1,4,7-triacetic acid) (NOTA) has been evaluated as the five-coordinate 64Cu complex, with results indicating GRPR-specific tumor uptake. This study aimed to conjugate S-2-(4-isothiocyanatobenzyl)-NOTA (p-SCN-Bn-NOTA) to BN(7-14) such that it could form a six-coordinate complex with 64Cu and to evaluate the resulting peptide. METHODS: p-SCN-NOTA was conjugated to 8-aminooctanoic acid (Aoc)-BN(7-14) in solution to yield NOTA-Bn-SCN-Aoc-BN(7-14). The unlabeled peptide was evaluated in a cell binding assay using PC-3 prostate cancer cells and 125I-Tyr4-BN to determine the IC50 value. The peptide was radiolabeled with 64Cu and evaluated for internalization into PC-3 cells and for tumor uptake in mice bearing PC-3 xenografts using biodistribution and micro-positron emission tomography imaging studies. RESULTS: The binding assay demonstrated that NOTA-Bn-SCN-Aoc-BN(7-14) bound with high affinity to GRPR with an IC50 of 1.4 nM. The radiolabeled peptide demonstrated time-dependent internalization into PC-3 cells. In vivo, the peptide demonstrated tumor-specific uptake and imaging that were comparable to those of previously reported 64Cu-labeled BN analogues. CONCLUSIONS: These studies demonstrate that 64Cu-NOTA-Bn-SCN-Aoc-BN(7-14) binds to GRPR-expressing cells and that it can be used for imaging of GRPR-expressing prostate cancer.

Towards novel radiosensitizing agents: the role of cytosolic PLA2α in combined modality cancer therapy.

The radioresistant nature of some tumors serves as an obstacle to curative therapy for several poor-prognosis malignancies. The radiosensitivity of a cancer is dependent not only on the intrinsic ability of tumor cells to recover from radiation-induced damage, but also the ability of stromal elements (e.g., vasculature) in the tumor microenvironment to survive and continue proliferating in the face of ionizing radiation. In this regard, it is important to understand the initial events activating radiation-induced signal transduction pathways. Among these events is the activation of cytosolic phospholipase A2 α and the subsequent production of the lipid second messengers. These events occur within minutes following exposure to ionizing radiation, and have been shown to enhance cell viability through a number of prosurvival signaling pathways. Furthermore, inhibition of cytosolic phospholipase A2 α has now been shown to reduce the viability of endothelial cells in culture after exposure to ionizing radiation, as well as slowing the growth of tumors in animal models of cancer.

A bioinformatics approach for biomarker identification in radiation-induced lung inflammation from limited proteomics data.

Many efforts have been made to discover novel bio-markers for early disease detection in oncology. However, the lack of efficient computational strategies impedes the discovery of disease-specific biomarkers for better understanding and management of treatment outcomes. In this study, we propose a novel graph-based scoring function to rank and identify the most robust biomarkers from limited proteomics data. The proposed method measures the proximity between candidate proteins identified by mass spectrometry (MS) analysis utilizing prior reported knowledge in the literature. Recent advances in mass spectrometry provide new opportunities to identify unique biomarkers from peripheral blood samples in complex treatment modalities such as radiation therapy (radiotherapy), which enables early disease detection, disease progression monitoring, and targeted intervention. Specifically, the dose-limiting role of radiation-induced lung injury known as radiation pneumonitis (RP) in lung cancer patients receiving radiotherapy motivates the search for robust predictive biomarkers. In this case study, plasma from 26 locally advanced non-small cell lung cancer (NSCLC) patients treated with radiotherapy in a longitudinal 3 × 3 matched-control cohort was fractionated using in-line, sequential multiaffinity chromatography. The complex peptide mixtures from endoprotease digestions were analyzed using comparative, high-resolution liquid chromatography (LC)-MS to identify and quantify differential peptide signals. Through analysis of survey mass spectra and annotations of peptides from the tandem spectra, we found candidate proteins that appear to be associated with RP. On the basis of the proposed methodology, α-2-macroglobulin (α2M) was unambiguously ranked as the top candidate protein. As independent validation of this candidate protein, enzyme-linked immunosorbent assay (ELISA) experiments were performed on independent cohort of 20 patients' samples resulting in early significant discrimination between RP and non-RP patients (p = 0.002). These results suggest that the proposed methodology based on longitudinal proteomics analysis and a novel bioinformatics ranking algorithm is a potentially promising approach for the challenging problem of identifying relevant biomarkers in sample-limited clinical applications.

A novel p38 alpha MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model.

BACKGROUND: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. METHODS: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. RESULTS: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. CONCLUSION: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Glia as a therapeutic target: selective suppression of human amyloid-beta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration.

A corollary of the neuroinflammation hypothesis is that selective suppression of neurotoxic products produced by excessive glial activation will result in neuroprotection. We report here that daily oral administration to mice of the brain-penetrant compound 4,6-diphenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (MW01-5-188WH), a selective inhibitor of proinflammatory cytokine production by activated glia, suppressed the human amyloid-beta (Abeta) 1-42-induced upregulation of interleukin-1beta, tumor necrosis factor-alpha, and S100B in the hippocampus. Suppression of neuroinflammation was accompanied by restoration of hippocampal synaptic dysfunction markers synaptophysin and postsynaptic density-95 back toward control levels. Consistent with the neuropathophysiological improvements, MW01-5-188WH therapy attenuated deficits in Y maze behavior, a hippocampal-linked task. Oral MW01-5-188WH therapy begun 3 weeks after initiation of intracerebroventricular infusion of human Abeta decreased the numbers of activated astrocytes and microglia and the cytokine levels in the hippocampus without modifying amyloid plaque burden or altering peripheral tissue cytokine upregulation in response to an in vivo inflammatory challenge. The results provide a novel integrative chemical biology proof in support of the neuroinflammation hypothesis of disease progression, demonstrate that neurodegeneration can be attenuated independently of plaque modulation by targeting innate brain proinflammatory cytokine responses, and indicate the feasibility of developing efficacious, safe, and selective therapies for neurodegenerative disorders by targeting key glial activation pathways.

Human amyloid beta-induced neuroinflammation is an early event in neurodegeneration.

Using a human amyloid beta (Abeta) intracerebroventricular infusion mouse model of Alzheimer's disease-related injury, we previously demonstrated that systemic administration of a glial activation inhibitor could suppress neuroinflammation, prevent synaptic damage, and attenuate hippocampal-dependent behavioral deficits. We report that Abeta-induced neuroinflammation is an early event associated with onset and progression of pathophysiology, can be suppressed by the glial inhibitor over a range of intervention start times, and is amenable to suppression without inhibiting peripheral tissue inflammatory responses. Specifically, hippocampal neuroinflammation and neurodegeneration occur in close time proximity at 4-6 weeks after the start of infusion. Intraperitoneal administration of inhibitor for 2-week intervals starting at various times after initiation of Abeta infusion suppresses progression of pathophysiology. The glial inhibitor is a selective suppressor of neuroinflammation, in that it does not block peripheral tissue production of proinflammatory cytokines or markers of B- and T-cell activation after a systemic lipopolysaccharide challenge. These results support a causal link between neuroinflammation and neurodegeneration, have important implications for future therapeutic development, and provide insight into the relative time window for targeting neuroinflammation with positive neurological outcomes.

Neuroinflammation: a potential therapeutic target.

The increased appreciation of the importance of glial cell-propagated inflammation (termed 'neuroinflammation') in the progression of pathophysiology for diverse neurodegenerative diseases, has heightened interest in the rapid discovery of neuroinflammation-targeted therapeutics. Efforts include searches among existing drugs approved for other uses, as well as development of novel synthetic compounds that selectively downregulate neuroinflammatory responses. The use of existing drugs to target neuroinflammation has largely met with failure due to lack of efficacy or untoward side effects. However, the de novo development of new classes of therapeutics based on targeting selective aspects of glia activation pathways and glia-mediated pathophysiologies, versus targeting pathways of quantitative importance in non-CNS inflammatory responses, is yielding promising results in preclinical animal models. The authors briefly review selected clinical and preclinical data that reflect the prevailing approaches targeting neuroinflammation as a pathophysiological process contributing to onset or progression of neurodegenerative diseases. The authors conclude with opinions based on recent experimental proofs of concept using preclinical animal models of pathophysiology. The focus is on Alzheimer's disease, but the concepts are transferrable to other neurodegenerative disorders with an inflammatory component.

Validation of the neuroinflammation cycle as a drug discovery target using integrative chemical biology and lead compound development with an Alzheimer's disease-related mouse model.

The neuroinflammation cycle has been proposed as a potential therapeutic target in the development of new approaches to altering Alzheimer's disease (AD) progression. However, the efficacy and toxicological profile of compounds that focus only on classical NSAID targets have been disappointing to date. Therefore, we recently initiated an unbiased, integrative chemical biology approach that used a hierarchal set of cell-based screens, followed by efficacy analysis in a new AD-relevant animal model that more closely resembles human pathology endpoints in terms of neuroinflammation and neuronal loss. The prior investigations provided a proof of concept that targeting the neuroinflammation cycle may be a viable drug discovery approach for AD. However, recent informatics analyses of the high attrition rate in drug development have identified the need for starting drug development with lead compounds that are well below cut off values in computed molecular properties in order to facilitate late stage medicinal chemistry refinement to improve in vivo functions. We describe here how we are leveraging our novel, unbiased, integrative chemical biology approach for the rapid discovery of potential lead compounds for AD drug discovery. Specifically, we show that orally bioavailable compounds with the desired physical properties and in vivo functions can be identified in focused synthetic libraries composed of chemical diversifications of the inactive but privileged pyridazine molecular fragment.

Interleukin 1 receptor antagonist knockout mice show enhanced microglial activation and neuronal damage induced by intracerebroventricular infusion of human beta-amyloid.

BACKGROUND: Interleukin 1 (IL-1) is a key mediator of immune responses in health and disease. Although classically the function of IL-1 has been studied in the systemic immune system, research in the past decade has revealed analogous roles in the CNS where the cytokine can contribute to the neuroinflammation and neuropathology seen in a number of neurodegenerative diseases. In Alzheimer's disease (AD), for example, pre-clinical and clinical studies have implicated IL-1 in the progression of a pathologic, glia-mediated pro-inflammatory state in the CNS. The glia-driven neuroinflammation can lead to neuronal damage, which, in turn, stimulates further glia activation, potentially propagating a detrimental cycle that contributes to progression of pathology. A prediction of this neuroinflammation hypothesis is that increased IL-1 signaling in vivo would correlate with increased severity of AD-relevant neuroinflammation and neuronal damage. METHODS: To test the hypothesis that increased IL-1 signaling predisposes animals to beta-amyloid (Abeta)-induced damage, we used IL-1 receptor antagonist Knock-Out (IL1raKO) and wild-type (WT) littermate mice in a model that involves intracerebroventricular infusion of human oligomeric Abeta1-42. This model mimics many features of AD, including robust neuroinflammation, Abeta plaques, synaptic damage and neuronal loss in the hippocampus. IL1raKO and WT mice were infused with Abeta for 28 days, sacrificed at 42 days, and hippocampal endpoints analyzed. RESULTS: IL1raKO mice showed increased vulnerability to Abeta-induced neuropathology relative to their WT counterparts. Specifically, IL1raKO mice exhibited increased mortality, enhanced microglial activation and neuroinflammation, and more pronounced loss of synaptic markers. Interestingly, Abeta-induced astrocyte responses were not significantly different between WT and IL1raKO mice, suggesting that enhanced IL-1 signaling predominately affects microglia. CONCLUSION: Our data are consistent with the neuroinflammation hypothesis whereby increased IL-1 signaling in AD enhances glia activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.

Enhanced susceptibility of S-100B transgenic mice to neuroinflammation and neuronal dysfunction induced by intracerebroventricular infusion of human beta-amyloid.

S-100B is an astrocyte-derived protein that is increased in focal areas of the brain most severely affected by neuropathological changes in Alzheimer's disease (AD). Cell-based and clinical studies have implicated S-100B in progression of a pathologic, glial-mediated pro-inflammatory state in the CNS. However, the relationship between S-100B levels and susceptibility to AD-relevant neuroinflammation and neuronal dysfunction in vivo has not been determined. To test the hypothesis that overexpression of S-100B increases vulnerability to beta-amyloid (Abeta)-induced damage, we used S-100B-overexpressing transgenic (Tg) and S-100B knockout (KO) mice in a mouse model that involves intracerebroventricular infusion of human oligomeric Abeta1-42. This model mimics many features of AD, including robust neuroinflammation, Abeta plaques, synaptic damage and neuronal loss in the hippocampus. S-100B Tg, KO, and wild-type (WT) mice were infused with Abeta for 28 days, sacrificed at 60 days, and hippocampal endpoints analyzed. We found that Tg mice showed increased vulnerability to Abeta-induced neuropathology relative to either WT or KO mice. Specifically, Tg mice exhibited enhanced glial activation and neuroinflammation, increased nitrotyrosine staining (a marker of glial-induced neuronal damage), and more pronounced loss of synaptic markers. Interestingly, Tg mice showed no significant differences in Abeta plaque burden compared with WT or KO mice, suggesting that, as in the human situation, the severity of neuronal dysfunction did not correlate with amyloid deposition. Our data are consistent with a model in which S-100B overexpression in AD enhances glial activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.

Aminopyridazines inhibit beta-amyloid-induced glial activation and neuronal damage in vivo.

The critical role of chronic inflammation in disease progression continues to be increasingly appreciated across multiple disease areas, especially in neurodegenerative disorders such as Alzheimer's disease. We report that late intervention with a recently discovered aminopyridazine suppressor of glial activation, developed to inhibit both oxidative and inflammatory cytokine pathways, attenuates human amyloid beta (Abeta)-induced glial activation in a murine model. Peripheral administration of the aminopyridazine MW01-070C, beginning 3 weeks after the start of intracerebroventricular infusion of human Abeta1-42, decreased the number of activated astrocytes and microglia and the levels of proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha and S100B in the hippocampus. Inhibition of neuroinflammation correlated with a decreased neuron loss, restoration towards control levels of synaptic dysfunction biomarkers in the hippocampus, and diminished amyloid plaque deposition. The results from this in vivo chemical biology approach provide a proof of concept that targeting of key glia inflammatory cytokine pathways can suppress Abeta-induced neuroinflammation in vivo, with resultant attenuation of neuronal damage.

Aminopyridazines attenuate hippocampus-dependent behavioral deficits induced by human beta-amyloid in a murine model of neuroinflammation.

The importance of glial cell-driven neuroinflammation in the pathogenesis and progression of Alzheimer's disease (AD) led us to initiate a drug discovery effort targeting the neuroinflammatory cycle that is characteristic of AD. We used our synthetic chemistry platform focused on bioavailable aminopyridazines as a new chemotype for AD drug discovery to develop novel, selective suppressors of key inflammatory and oxidative pathways in glia. We found that MW01-070C, an aminopyridazine that works via mechanisms distinct from NSAIDs and p38 MAPK inhibitors, attenuates beta-amyloid (Abeta)-induced neuroinflammation and neuronal dysfunction in a dose-dependent manner, and prevents Abeta-induced behavioral impairment. In vivo data were obtained with a murine model that uses intraventricular infusion of human Abeta1-42 peptide and replicates many of the hallmarks of AD pathology, including neuroinflammation, neuronal and synaptic degeneration, and amyloid deposition. The quantifiable endpoint pathology is robust, reproducible, and rapid in onset. Our results provide a proof of concept that targeting neuroinflammation with aminopyridazines is a viable AD drug discovery approach that has the potential to modulate disease progression and document the utility of this mouse model for preclinical screening of compounds targeting AD-relevant neuroinflammation and neuronal death.

Increased susceptibility of S100B transgenic mice to perinatal hypoxia-ischemia.

S100B is a glial-derived protein that is a well-established biomarker for severity of neurological injury and prognosis for recovery. Cell-based and clinical studies have implicated S100B in the initiation and maintenance of a pathological, glial-mediated proinflammatory state in the central nervous system. However, the relationship between S100B levels and susceptibility to neurological injury in vivo has not been determined. We used S100B transgenic (Tg) and knockout (KO) mice to test the hypothesis that overexpression of S100B increases vulnerability to cerebral hypoxic-ischemic injury and that this response correlates with an increase in neuroinflammation from activated glia. Postnatal day 8 Tg mice subjected to hypoxia-ischemia showed a significant increase in mortality compared with KO and wild-type mice. Tg mice also exhibited greater cerebral injury and volume loss in the ischemic hemisphere after an 8-day recovery, as assessed by histopathology and magnetic resonance imaging. Measurement of glial fibrillary acidic protein and S100B levels showed a significant increase in the Tg mice, consistent with heightened glial activation and neuroinflammation in response to injury. This is the first demonstration to our knowledge that overexpression of S100B in vivo enhances pathological response to injury.

Discovery of a new class of synthetic protein kinase inhibitors that suppress selective aspects of glial activation and protect against beta-amyloid induced injury: a foundation for future medicinal chemistry efforts focused on targeting Alzheimer's disease progression.

A prevailing hypothesis in Alzheimer's disease (AD) research is that chronically activated glia may contribute to neuronal dysfunction, through generation of a detrimental state of neuroinflammation. This raises the possibility in drug discovery research of targeting the cycle of untoward glial activation and neuronal dysfunction that characterizes neuroinflammation. Success over the past century with effective anti-inflammatory drug development, in which the molecular targets are intracellular enzymes involved in signal transduction events and cellular homeostasis, demands that a similar approach be tried with neuroinflammation. Suggestive clinical correlations between inflammation markers and AD contribute to the urgency in addressing the hypothesis that targeting selective glial activation processes might be a therapeutic approach complementary to existing drugs and discovery efforts. An academic collaboratorium initiated a rapid inhibitor discovery effort 2 yr ago, focused on development of novel compounds with new mechanisms of action in AD-relevant cellular processes, in order to obtain the small-molecule compounds required to address the neuroinflammation hypothesis and provide a proof of concept for future medicinal chemistry efforts. We summarize here our progress toward this goal in which novel pyridazine-based inhibitors of gene-regulating protein kinases have been discovered. Feasibility studies indicate their potential utility in current medicinal chemistry efforts focused on improvement in molecular properties and the longer term targeting of AD-related pathogenic processes.