Development of an Alamar Blue (TM) Viability Assay in 384-Well Format for High Throughput Whole Cell Screening of Trypanosoma brucei brucei Bloodstream Form Strain 427
Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland, Australia.The American journal of tropical medicine and hygiene (Impact Factor: 2.7). 10/2009; 81(4):665-74. DOI: 10.4269/ajtmh.2009.09-0015
There is an urgent need for new compounds for the drug development pipeline for treatment of patients with African sleeping sickness. One approach for identifying such compounds is by high throughput screening (HTS) of compound collections. For time and cost considerations, there is a need for the development of an assay that uses at least 384-well formats. To our knowledge, there are currently no viability assays for whole cell screening of trypanosomes in the 384-well plate format. We have developed and optimized an Alamar Blue viability assay in a 384-well format for Trypanosoma brucei brucei bloodstream form strain 427 (BS427). The assay had a Z' > 0.5 and tolerated a final dimethyl-sulfoxide concentration of 0.42%. Drug sensitivity was compared with those reported from previously developed 96-well methods and was found to be comparable. The sensitivity and cost benefit of the Alamar Blue assay make it an excellent candidate for HTS application.
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- "T. b. brucei growth inhibition assays T. b. brucei 427 bloodstream trypanosomes were cultured in vitro at 37 C in 5% CO 2 with HM1-9 medium supplemented with 10% foetal bovine serum (FBS; Thermo Scientific). Compound activity was assessed using an Alamar blue ® viability assay, as previously described (Sykes and Avery, 2009). Briefly, logarithmic phase T. b. brucei parasites (1200 cells/ml) were added to 384-well microtitre plates and incubated for 24 h at 37 C in 5% CO 2 . "
ABSTRACT: Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with three HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat®), romidepsin (Istodax®) and belinostat (Beleodaq®), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10–200 nM), while only romidepsin was active at sub-μM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4.International Journal for Parasitology: Drugs and Drug Resistance 06/2015; 2012(3). DOI:10.1016/j.ijpddr.2015.05.004 · 3.29 Impact Factor
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- "When added to cells, the PrestoBlue TM reagent – containing a non-fluorescent, cell-permeant compound – is modified by the reducing environment of the viable cells, becoming highly fluorescent, which can be detected using fluorescence or absorbance measurements . PrestoBlue TM reagent is more sensitive than AlamarBlue ® , which is a redox indicator of enzyme activity widely used in whole organism screening  and is extensively used in screening tests of viability and cytotoxicity      . PrestoBlue TM was directly added to the cells in the culture medium at a final concentration of 10%. "
ABSTRACT: During therapeutic interventions, blood concentrations of intravenously applied drugs are higher, and their onset of pharmacological action is faster than with other routes of drug administration. However, acute drug therapy often produces nephrotoxic side effects, as commonly seen after treatment with Ketorolac or Gentamicin leading to questions about their use, especially for patients at risk for acute renal failure. Omega-6(n-6) and omega-3(n-3) polyunsaturated fatty acids (PUFA) affect eicosanoid metabolism, which plays a role in the regulation of inflammation. Eicosanoids derived from n-6 FA have proinflammatory and immunoactive functions, whereas eicosanoids derived from n-3 PUFA have anti-inflammatory and cytoprotective properties. We hypothesized that providing such injectable drugs with nephrotoxic potential in combination with n3-PUFAs from the outset, might afford rapid cytoprotection of renal cells, given the recent evidence that intravenously administered n3-PUFAs are rapidly incorporated into cell membranes. We used intraglomerular mesangial cells (MES13) that are sensitive to treatment with Ketorolac or Gentamicin instead of proximal tubular cells which do not respond to Ketorolac. We found a significant inhibition of Ketorolac (0.25, 0.5, 1 mM) or Gentamicin (2.5, 5 mM) induced cytotoxicity after pretreatment of MES13 cells with 0.01% of 20%w/v LipOmega-3 Emulsion 9/1, containing 90:10 wt/wt mixture of fish oil derived triglycerides to medium chain triglycerides.Toxicology Reports 10/2014; 1. DOI:10.1016/j.toxrep.2014.10.011
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- "The traditional route to identify such therapeutic agents has been to search for compounds that will target genes or pathways necessary for cell viability. Indeed, whole-cell based high-throughput screening for trypanostatic and trypanocidal agents is well established (Hesse et al., 1995; Mackey et al., 2006; Sykes and Avery, 2009; Jones et al., 2010; De Rycker et al., 2012; Sykes et al., 2012; Vodnala et al., 2013) and multiple classes of compounds with potential for development into new therapeutics are being pursued (Mackey et al., 2006; Jones et al., 2010; Sykes et al., 2012; Vodnala et al., 2013). However, phenotypic screens for genes or pathways involved in the establishment, maintenance or transmission of infection could provide an alternative or complementary route to the identification of novel therapeutics with anti-virulence or transmission-blocking potential. "
ABSTRACT: African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimize transmission and to prevent uncontrolled parasite multiplication overwhelming the host. In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signaling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed.Frontiers in Cellular and Infection Microbiology 11/2013; 3:78. DOI:10.3389/fcimb.2013.00078 · 3.72 Impact Factor
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