Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas

Sugar-Sweetened Beverage Intake and Cancer Recurrence and Survival in CALGB 89803 (Alliance)

Background: In colon cancer patients, obesity, sedentary lifestyle, and high dietary glycemic load have been associated with increased risk of cancer recurrence. High sugar-sweetened beverage intake has been associated with obesity, diabetes, and cardio-metabolic diseases, but the influence on colon cancer survival is unknown. Methods: We assessed the association between sugar-sweetened beverage consumption on cancer recurrence and mortality in 1,011 stage III colon cancer patients who completed food frequency questionnaires as part of a U.S. National Cancer Institute-sponsored adjuvant chemotherapy trial. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated with Cox proportional hazard models. Results: Patients consuming ≥2 servings of sugar-sweetened beverages per day experienced an adjusted HR for disease recurrence or mortality of 1.67 (95% CI, 1.04–2.68), compared with those consuming <2 servings per month (Ptrend = 0.02). The association of sugar-sweetened beverages on cancer recurrence or mortality appeared greater among patients who were both overweight (body mass index ≥25 kg/m2) and less physically active (metabolic equivalent task-hours per week <18) (HR = 2.22; 95% CI, 1.29–3.81, Ptrend = 0.0025). Conclusion: Higher sugar-sweetened beverage intake was associated with a significantly increased risk of cancer recurrence and mortality in stage III colon cancer patients

Estimating Grizzly and Black Bear Population Abundance and Trend in Banff National Park Using Noninvasive Genetic Sampling

We evaluated the potential of two noninvasive genetic sampling methods, hair traps and bear rub surveys, to estimate population abundance and trend of grizzly (Ursus arctos) and black bear (U. americanus) populations in Banff National Park, Alberta, Canada. Using Huggins closed population mark-recapture models, we obtained the first precise abundance estimates for grizzly bears ( = 73.5, 95% CI = 64–94 in 2006;  = 50.4, 95% CI = 49–59 in 2008) and black bears ( = 62.6, 95% CI = 51–89 in 2006;  = 81.8, 95% CI = 72–102 in 2008) in the Bow Valley. Hair traps had high detection rates for female grizzlies, and male and female black bears, but extremely low detection rates for male grizzlies. Conversely, bear rubs had high detection rates for male and female grizzlies, but low rates for black bears. We estimated realized population growth rates, lambda, for grizzly bear males ( = 0.93, 95% CI = 0.74–1.17) and females ( = 0.90, 95% CI = 0.67–1.20) using Pradel open population models with three years of bear rub data. Lambda estimates are supported by abundance estimates from combined hair trap/bear rub closed population models and are consistent with a system that is likely driven by high levels of human-caused mortality. Our results suggest that bear rub surveys would provide an efficient and powerful means to inventory and monitor grizzly bear populations in the Central Canadian Rocky Mountains

Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease

Genetic association studies have identified 215 risk loci for inflammatory bowel disease, thereby uncovering fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals and conducted a meta-analysis with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new susceptibility loci, 3 of which contain integrin genes that encode proteins in pathways that have been identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes ($\textit{ITGA4 }$ and $\textit{ITGB8}$) and at previously implicated loci ($\textit{ITGAL }$and $\textit{ICAM1}$). In all four cases, the expression-increasing allele also increases disease risk. We also identified likely causal missense variants in a gene implicated in primary immune deficiency, $\textit{PLCG2}$, and a negative regulator of inflammation, $\textit{SLAMF8}$. Our results demonstrate that new associations at common variants continue to identify genes relevant to therapeutic target identification and prioritization.This work was co-funded by the Wellcome Trust [098051] and the Medical Research Council, UK [MR/J00314X/1]. Case collections were supported by Crohn’s and Colitis UK. KMdL, LM, CAL, YL, DR, JG-A, NJP, CAA and JCB are supported by the Wellcome Trust [098051; 093885/Z/10/Z; 094491/Z/10/Z]. KMdL is supported by a Woolf Fisher Trust scholarship. CAL is a clinical lecturer funded by the NIHR. We thank Anna Stanton for co-ordinating the Guy’s and St Thomas’ patient recruitment. We acknowledge support from the Department of Health via the NIHR comprehensive Biomedical Research Centre awards to Guy’s and St Thomas’ NHS Foundation Trust in partnership with King’s College London and to Addenbrooke’s Hospital, Cambridge in partnership with the University of Cambridge. This research was also supported by the NIHR Newcastle Biomedical Research Centre. The UK Household Longitudinal Study is led by the Institute for Social and Economic Research at the University of Essex and funded by the Economic and Social Research Council

HLA-DQA1*05 carriage associated with development of anti-drug antibodies to infliximab and adalimumab in patients with Crohn's Disease

Anti-tumor necrosis factor (anti-TNF) therapies are the most widely used biologic drugs for treating immune-mediated diseases, but repeated administration can induce the formation of anti-drug antibodies. The ability to identify patients at increased risk for development of anti-drug antibodies would facilitate selection of therapy and use of preventative strategies.This article is freely available via Open Access. Click on Publisher URL to access the full-text

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

CSF1 Restores Innate Immunity Following Liver Injury in Mice and Serum Levels Indicate Outcomes of Patients With Acute Liver Failure

Background &#38; Aims: Liver regeneration requires functional liver macrophages, which provide an immune barrier that is compromised after liver injury. The numbers of liver macrophages are controlled by macrophage colony-stimulating factor (CSF1). We examined the prognostic significance of the serum level of CSF1 in patients with acute liver injury and studied its effects in mice. Methods: We measured levels of CSF1 in serum samples collected from 55 patients who underwent partial hepatectomy at the Royal Infirmary Edinburgh between December 2012 and October 2013, as well as from 78 patients with acetaminophen-induced acute liver failure admitted to the Royal Infirmary Edinburgh or the University of Kansas Medical Centre. We studied the effects of increased levels of CSF1 in uninjured mice that express wild-type CSF1 receptor or a constitutive or inducible CSF1-receptor reporter, as well as in chemokine receptor 2 (Ccr2)-/- mice; we performed fate-tracing experiments using bone marrow chimeras. We administered CSF1-Fc (fragment, crystallizable) to mice after partial hepatectomy and acetaminophen intoxication, and measured regenerative parameters and innate immunity by clearance of fluorescent microbeads and bacterial particles. Results: Serum levels of CSF1 increased in patients undergoing liver surgery in proportion to the extent of liver resected. In patients with acetaminophen-induced acute liver failure, a low serum level of CSF1 was associated with increased mortality. In mice, administration of CSF1-Fc promoted hepatic macrophage accumulation via proliferation of resident macrophages and recruitment of monocytes. CSF1-Fc also promoted transdifferentiation of infiltrating monocytes into cells with a hepatic macrophage phenotype. CSF1-Fc increased innate immunity in mice after partial hepatectomy or acetaminophen-induced injury, with resident hepatic macrophage as the main effector cells. Conclusions: Serum CSF1 appears to be a prognostic marker for patients with acute liver injury. CSF1 might be developed as a therapeutic agent to restore innate immune function after liver injury

Inherited determinants of Crohn's disease and ulcerative colitis phenotypes: a genetic association study

Crohn's disease and ulcerative colitis are the two major forms of inflammatory bowel disease; treatment strategies have historically been determined by this binary categorisation. Genetic studies have identified 163 susceptibility loci for inflammatory bowel disease, mostly shared between Crohn's disease and ulcerative colitis. We undertook the largest genotype association study, to date, in widely used clinical subphenotypes of inflammatory bowel disease with the goal of further understanding the biological relations between diseases

Studies towards the total synthesis of eleutherobin and other marine natural products

The primary focus of the research described in this document relates to the development and application of new synthetic methodologies relevant for the concise construction of four natural products. In Chapter 2, a discussion of our investigation of the total synthesis of eleutherobin (1) is disclosed. Eleutherobin (1), first isolated in 1997 from the rare soft coral Eleutherobia sp., is a member of a class of microtubule stabilising natural products. Although it displays potent cytotoxicity, its development as an anticancer drug has been hampered by the scarcity of material available from the natural source. In an effort to produce quantities of eleutherobin required for further biological testing, four conceptually unique approaches to eleutherobin were investigated which culminated in the development of an unprecedented palladium-catalysed α-arylation reaction/Friedel-Crafts cyclisation methodology for tetralone synthesis. This strategy permitted the production of multi-gram quantities of an advanced tetralone intermediate, and enabled the synthesis of a functionalised epoxyenone intermediate along our intended synthetic route. These investigations have provided a solid foundation for an eventual synthesis of eleutherobin that may also facilitate the evaluation of this natural product as an anticancer drug. In Chapter 3, the total synthesis of two potent anthelmintic oxylipid natural products, isolated from Notheia anomala, is discussed. Specifically, a silver-mediated cyclisation of two chlorodiols afforded two diastereomeric styryl-tetrahydrofurans, which were rapidly elaborated into the desired natural products. In addition, these syntheses featured a remarkable example of inverse-temperature dependence in the diastereoselective addition of Grignard reagents to tetrahydrofurfurals. Ultimately, these natural products were prepared in six synthetic transformations in excellent overall yield and efficiency. The last two topics presented in this thesis are contained in two separate appendices and highlight our interest in the synthesis of ecologically relevant natural products. In Appendix A, we report the synthesis and structure determination of the unknown banana volatile, (3R,2’S)-(2’-pentyl)-3-hydroxyhexanoate, and its olfactory recognition by the common fruit fly. The work presented in Appendix B focuses on the development of a scalable synthesis of mathuralure, the sex pheromone of the pink gypsy moth, Lymantria mathura, a potentially devastating invasive species

Development of liposomal bupivacaine using a transmembrane pH gradient

There is a clear clinical requirement for longer acting local anaesthetics, particularly for the management of post-operative and chronic pain. In this regard, liposomes have been suggested to represent a potentially useful vehicle for sustained drug release following local administration. In this thesis, a transmembrane pH gradient was employed to efficiently encapsulate bupivacaine within large unilamellar vesicles. The rate and extent of bupivacaine uptake into large unilamellar vesicles exhibiting a pH gradient (interior acidic) were determined and compared to drug association with control liposomes that did not exhibit a proton gradient. Subsequent studies examined the kinetics of bupivacaine release from the liposome systems in vitro. Using the Guinea Pig cutaneous wheal model, the rate of clearance of the liposome carrier was monitored following intradermal administration employing a radiolabeled lipid marker and the duration of nerve blockade produced by free and liposomal bupivacaine compared. Intraperitoneal injections of bupivacaine encapsulated in pH gradient vesicles, control (no pH gradient vesicles) and free drug were completed in mice to determine the relative toxicities. While bupivacaine is rapidly and efficiently accumulated within liposomes exhibiting a pH gradient (interior acidic), little uptake was seen for control vesicles. Using an in vitro model of drug clearance, liposomally encapsulated bupivacaine was found to be released more slowly and over a longer period of time compared to either the free drug or bupivacaine associated with control (no pH gradient liposomes). In the Guinea Pig cutaneous wheal model, over 85% of the liposomal carrier was found to remain at the site of administration over two days and the sustained drug release afforded by liposomes exhibiting a pH gradient resulted in a threefold increase in the duration of nerve blockade compared to either the free drug or bupivacaine in the presence of control (no pH gradient) liposomes. In the toxicity study, bupivacaine encapsulated in pH gradient vesicles showed a greater than fourfold increase in safety compared to the use of the free drug and control (no pH gradient) vesicles. The present results clearly establish that large unilamellar vesicles exhibiting a pH gradient can efficiently encapsulate bupivacaine and subsequently provide a sustained release system that greatly increases the duration of neural blockade and, in addition, reduces the toxicity.Pharmaceutical Sciences, Faculty ofGraduat