Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade
2008

A Simeonov
A Jadhav
A A Sayed
Y Wang
M E Nelson
Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

PLoS Negl Trop Dis
211272008Received: August 9, 2007; Accepted: October 16, 2007; Published: January 2, 2008Editor: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7-to 15-point concentration series at 5 mL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC 50 s ranging from micromolar to the assay response limit (,25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases.This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

Introduction

Schistosomiasis, also known as bilharzia, a debilitating disease resulting from the infection by the trematode parasite Schistosoma ssp. (S. mansoni, S. mekongi, S. japonicum, S. haematobium, and S. intercalatum) currently affects over 200 million people worldwide, mostly in developing countries [1]. A large percentage of those infected exhibit severe morbidity manifested as growth stunting, lassitude, and cognitive impairment [2], and an estimated 280,000 people die annually from the disease in sub-Saharan Africa alone [3]. The primary route of infection is via unsafe river and lake water, which is widely used in sub-Saharan Africa and Southeast Asia, among other regions, for irrigation, drinking, cooking, and bathing. Larval parasite forms (residing in and released by snails) can penetrate the skin of people contacting the water. The lifecycle of Schistosoma is exceedingly complex, with the parasite going through a number of stages both outside and inside the human host. Once inside humans, it can survive for years, even decades [4].

The need to control schistosomiasis is acute and efforts have been ongoing for years on three main fronts: prevention (via establishment and maintenance of sources of safe potable water), development of a vaccine, and use of drugs to treat the infection [1]. Although the number of schistosomiasis cases worldwide is indeed stunning, the number of drugs available to treat the disease is surprisingly small. Earlier in the 20 th century, schistosomiasis was treated with highly toxic antimonial compounds, of which the most common was potassium antimonyl tartrate (PAT, tartar emetic). During the past three decades the only drug used against the infection is praziquantel, which is administered orally, is stable, effective against all major schistosome species in a single dose, and relatively inexpensive [5,6]. However, because of high reinfection rates, praziquantel must be administered on an annual or semiannual basis. While its exact mechanism of action is unclear, praziquantel is thought to affect the parasites by disrupting calcium homeostasis [7,8]. Preliminary reports of praziquantelresistant cases, and the generation of praziquantel-resistant parasites in the laboratory [9][10][11] highlight the need for new drugs to treat the disease. Artemisinin has shown promise as a new drug for schistosomiasis [12] although its use for schistosomiasis may be restricted in areas of malaria transmission so that its use as an antimalarial is not put at risk. Simplified derivatives of artemisinin, the 1,2,4-trioxolanes, show promise and potential selectivity, but these, like the parent compound, are significantly less active against adult schistosome parasites [13]. Oxamniquine, used extensively in Brazil in the past, is effective only against S. mansoni and resistance has been reported further reducing its potential value in schistosomiasis control [10].

Studies of the schistosome life cycle have focused on the fact it can survive for decades in the blood stream of the human host without being severely affected by the immune system and the associated assault by various reactive oxygen species (ROS). Since schistosomes do not have catalase to degrade hydrogen peroxide [14], other mechanisms must exist within the parasite to degrade ROS. Two uniquely positioned S. mansoni enzymes have been recently described that seem to act in concert to provide an effective antioxidant ''firewall''. Thioredoxin glutathione reductase (TGR) is a multifunctional selenocysteine-containing enzyme that catalyzes the interconversion between reduced and oxidized forms of both glutathione (GSH) and thioredoxin (Trx), which are major contributors to the maintenance of redox balance in eukaryotes [15]. In contrast, humans possess two distinct enzymes, glutathione reductase (GR) and thioredoxin reductase (TrxR), which specifically recognize GSH and Trx as substrates, respectively [16]. The apparent replacement of two human enzymes by one dual-specificity worm enzyme has created a metabolic and regulatory bottleneck in which the inactivation of a single target, TGR, might have an enhanced deleterious effect on both the maintenance of parasite's redox balance and on its ''antioxidant firewall''. Indeed, recent small molecule inhibition and RNA interference experiments have shown that inactivation of TGR has profound effects on S. mansoni survival rates both in culture and in infected mice [17]. Another component of the S. mansoni ''firewall'' are the peroxiredoxins (Prx), which are responsible for catalyzing the electron transfer to the main ROS agent hydrogen peroxide and, uniquely for schistosomes, from both GSH and Trx [18]. Thus, when TGR and Prx operate in concert, NADPH reducing equivalents are essentially transferred via TGR-catalyzed reaction to the oxidized forms of either Trx or GSH, while Trx or GSH in turn transfer reducing equivalents to hydrogen peroxide via Prx-catalyzed reactions ( Figure 1).

Such improved understanding of the organisms responsible for neglected tropical diseases (NTDs) presents opportunities for new drug development. However, private-sector biopharmaceutical interest in NTDs has traditionally been limited due to high risk and low expected return-on-investment of these projects, though this is beginning to change with the advent of increased philanthropic and public-private-government partnership funding [19]. A significant problem that remains, however, is the significant gap in technologies, expertise, and cultures between academic and biopharmaceutical organizations [20]. At the US National Institutes of Health (NIH), the NIH Roadmap Molecular Libraries Initiative (MLI) was started in 2004 in part to address this problem. The MLI provides academic investigators with the pharma-scale infrastructure and technologies necessary to discover both chemical probes of physiology, and starting points for development of novel therapeutics for the rare and neglected diseases that are of less interest to the pharmaceutical sector [21].

The TGR/Prx work described here is the result of the first project officially accepted by the MLI in 2005. Since inhibiting either TGR or Prx can potentially lead to schistosome death [22], we chose to screen both enzymes in one assay as a reconstituted redox cascade. While TGR and Prx2 can be assayed individually, the separate assays are relatively less robust. TGR can be assayed in a relatively simple colorimetric assay by following the catalytic reduction of DTNB (5,59 dithiobis(2-nitrobenzoic acid), Ellman's reagent) by NADPH; Prx2 at present can only be assayed with thioredoxin as a substrate together with TGR or thioredoxin reductase, or in a coupled reaction involving yeast glutathione reductase, and HTScompatible assays [23] have yet to be developed [17,18,24]. By performing the high-throughput screen against both enzymes (present at equivalent levels in the assay), we were able to address both novel targets simultaneously while also combining target deconvolution and confirmation at the post-screen stage. In this report, we describe the miniaturization to 1536-well density of a cuvette-based assay for the TGR/Prx2 cascade which utilizes as a quantitative measure the decrease in fluorescence of the consumed NADPH substrate, the performance of a quantitative highthroughput screen (qHTS) [25] against 71,028 discrete compounds, and the initial characterization of several novel series of inhibitors. The application of qHTS, in which each library compound is assayed at a range of concentrations to generate a dose-response profile, facilitated triaging of actives for the purpose of structureactivity relationship (SAR) analysis and lead expansion.

Materials and Methods Reagents

Nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reduced form (GSH), hydrogen peroxide, Tween-20 and

Author Summary

Schistosomiasis, also known as bilharzia, is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system, thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx2), have been recently identified and validated as targets for anti-schistosomiasis drug development. In search of inhibitors of this critical redox cascade, we optimized and performed a highly miniaturized automated screen of 71,028 compounds arrayed as 7-to 15-point dilution sets. We identified novel structural series of TGR inhibitors, several of which are highly potent and should serve both as mechanistic tools for probing redox pathways in S. mansoni and as starting points for developing much-needed new treatments for schistosomiasis. The paradigm presented here effectively bridges the gap between academic target identification and the first steps of drug development, and should be applicable to a variety of other important neglected diseases.

potassium antimonyl tartrate (PAT) were procured from Sigma-Aldrich. DMSO Certified ACS Grade was from Fisher. The screening assay was performed in 100 mM phosphate buffer pH 7.4 containing 10 mM EDTA and 0.01% Tween-20.

Preparation of Recombinant S. mansoni TGR and Prx2

Recombinant TGR with a fused bacterial-type SECIS element was expressed in the Escherichia coli strain BL21(DE3) (Invitrogen) in the presence of pSUABC in LB medium supplemented with 20 mM flavin adenine dinucleotide following conditions for optimal selenoprotein expression as described [17]. TGR was purified to homogeneity on an adenosine 29,59-diphosphate agarose (Sigma) column equilibrated with TE buffer as described [17]. TGR concentration was determined from the flavin adenine dinucleotide absorption (e 463 = 11.3 mM 21 cm 21 ). The pure protein was dialyzed against PBS and stored at 280uC.

Recombinant Prx2 in pRSETA was expressed in E. coli strain BLR(DE3)pLysS (Novagen) as described [18]. Briefly, after a 3-hr induction in 1 mM IPTG, cells were sonicated in 5% monothioglycerol (3-mercapto-1,2-propanediol) in 10 mM imidazole, 0.07 M Na 2 HPO 4 , 0.01 M NaH 2 PO 4 , and 0.15 M NaCl, pH 7.4. The supernatant was filtered and Prx2 was purified to homogeneity on a His Trap column (Amersham Biosciences). Protein purity was verified by SDS-PAGE. The purified protein was dialyzed against PBS and stored at 280uC until used.

Compound library

The 71,028 member library comprised two main subsets: 59,692 compounds from the NIH Molecular Libraries Small Molecule Repository (www.mli.nih.gov), prepared as 10 mM stock solutions in 384-well plates and delivered by Biofocus DPI (South San Francisco, CA, http://mlsmr.glpg.com/MLSMR_Home-Page/), and NCGC internal exploratory collection of approximately 11,336 compounds which consisted of several commercially available libraries of known bioactives (1280 compounds from Sigma-Aldrich (LOPAC1280 library), 1120 compounds from Prestwick Chemical Inc. (Washington, DC), 980 compounds from Tocris (Ellisville, Missouri), 280 purified natural products from TimTec (Newark, DE), 1980 compounds from the National Cancer Institute (the NCI Diversity Set)), 1408 National Institute of Environmental Health Sciences collection of known toxic compounds, as well as collections from other commercial and academic collaborators (three 1000-member combinatorial libraries from Pharmacopeia (Cranbury, NJ), 718 compounds from Boston University Center for Chemical Methodology and Library Development, 96-member peptide library from Prof. Sam Gelman's lab, University of Wisconsin, Madison, and 474 compounds from the University of Pittsburgh Center for Chemical Methodology and Library Development). The compound library (7 mL each in 1536well Greiner polypropylene compound plate) was prepared as DMSO solutions at initial concentrations ranging between 2 and 10 mM. Plate-to-plate (vertical) dilutions and 384-to-1536 compressions were performed on Evolution P3 dispense system equipped with 384-tip pipetting head and two RapidStak units (Perkin-Elmer, Wellesley, MA). Additional details on the preparation of the compound library are provided in Inglese et al [25].

Control plate

Titration of the known inhibitor PAT (PubChem CID6328158) was delivered via pin transfer from a separate plate to the lower half of column 2 of each assay plate. The starting concentration of the control, dissolved in 1:1 DMSO:water, was 1 mM, followed by five-fold dilution points in duplicate, for a total of eight concentrations.

qHTS protocol

Three mL of reagents (100 mM NADPH in columns 3 and 4 as negative control and 100 mM NADPH, 42 nM TGR, 700 mM GSH, 83 nM Prx2 mixture in columns 1, 2, 5-48) were dispensed into 1536-well Greiner black assay plates. Compounds and control (23 nL) were transferred via Kalypsys PinTool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, Palo Alto, CA). The plate was incubated for 15 min at room temperature, and then a 1 mL aliquot of 400 mM NADPH/700 mM GSH was added, immediately followed by a 1 mL aliquot of 2.5 mM H 2 O 2 to start the reaction. The plate was transferred to ViewLux highthroughput CCD imager (Perkin-Elmer, Wellesley, MA) where kinetic measurements (16 reads, one read every 30 sec) of the NADPH fluorescence decrease were acquired using 365 nm excitation/450 nm emission filter set. During dispense, the reagent bottles were kept submerged into 4uC recirculating chiller bath to minimize degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys, San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms (Staubli, Duncan, SC). Library plates were screened starting from the lowest and proceeding to the highest concentration. Vehicle-only plates, with DMSO being pin-transferred to the entire column 5-48 compound area, were inserted uniformly at the rate of approximately one plate for every 50 library plates in order to monitor for and record any shifts in the background.

Analysis of qHTS data

Time course data were collected on per-assay plate basis and were processed using in-house developed software. For each sample and at each individual concentration, 16 time points were processed using ordinary least squares regression to determine slope and intercept of linear fit. Additionally, a difference (delta) of last and first time point was generated for each time course. For activity calculations, delta values were chosen while the calculated slope, intercept, and the raw time-course data were stored in the database. Screening data were corrected and normalized and concentration-effect relationships derived by using the GeneData Screener software package (Basel, Switzerland). Percent activity was computed from the median values of the uninhibited, or neutral, control (48 wells located in column 1 and one-half of column 2) and the no-enzyme, or 100% inhibited, control (64 wells, entire columns 3 and 4), respectively. For assignment of plate concentrations and sample identifiers, ActivityBase (ID Business Solutions Ltd, Guildford, UK) was used for compound and plate registrations. An in-house database was used to track sample concentrations across plates. Correction factors were generated from the DMSO plate data and applied to each assay plate to correct for systematic errors in assay signal potentially resulting from issues with reagent dispensers or decrease in enzyme specific activity. A four parameter Hill equation [26] was fitted to the concentration-response data by minimizing the residual error between the modeled and observed responses. Outliers could be identified and masked by modeling the Hill equation and asking if the differences exceeded those expected from the noise in the assay.

The curve classification used is the same as described in Inglese et al. (2006) [25]. Briefly, concentration-response curves are placed into four classes: Class 1 contains complete concentration-response curves showing both upper and lower asymptotes and r 2 values .0.9. Class 2 contain incomplete concentration-response curves lacking the lower asymptote and show r 2 values .0.9. Class 3 curves are of the lowest confidence as they are defined by a single concentration point where the minimal acceptable activity is set at 3 SD of mean. Curves are classified as negative or positive depending on whether they exhibit signal decrease (apparent inhibition) or increase (apparent activation). Finally, Class 4 contains compounds that do not show any concentration response curves and are therefore classified as inactive.

A workflow was developed to facilitate a systematic approach in providing exhaustive analysis of structure activity relationships (SAR). First, a set of criteria used to define rules of determining an active set of compounds for the assay. These include decisions on inhibitors and activators, selectivity and counter screen information, curve class ranges, background fluorescence, etc. For this assay, compounds that showed signal activation (positive curve classes) were regarded as active due to fluorescence and were thus filtered out. The criteria are implemented as filters that are applied to rapidly define a core active set of compounds. This process eliminates many positive series that appear to have SAR and show reasonable titration response curves, but are not of biological relevance to the targets [23]. Next, the range of curve classes was limited to 21 through 23 to select for compounds showing signal decrease. Once an active set of compounds was identified, hierarchical agglomerative clustering with a 0.7 Tanimoto cutoff was performed using Leadscope (Leadscope Inc., Columbus, OH) fingerprints, which are ideally suited for two-dimensional scaffoldbased based clustering [27]. For each cluster, maximal common substructures (MCS) were extracted, and a manual step of trimming the MCSs was performed to create a list of scaffolds. This clustering step typically has overlapping compounds and thus can lead to overlapping MCSs. This list of trimmed scaffolds is abridged to a canonical set. Each scaffold is then represented as a precise definition to indicate number of attachments, ring size variability, etc. All filters were then relaxed to include all negative assay data. In the initial clustering, a set of singletons was found. These compounds were reported upon separately with their individual activity profiles. SAR series and singletons were finally ranked by their activity profile.

Follow-up testing of primary actives and analogs

Screening actives and analogues sourced as powders from the respective original suppliers (Sigma-Aldrich, NCI, Asinex, Chem Bridge, Tocris, Ambinter, and ChemDiv) were dissolved in DMSO to produce 10 mM initial stock solutions. The samples were then serially diluted row-wise in 384-well plate in twofold steps for a total of 24 concentrations, from 10 mM to 1.2 nM. Upon completion of the 24-point dilution, solutions from two 384well plates were transferred to duplicate wells of 1536-well compound plate. The last two rows of the 1536-well plate did not contain any test compound and were reserved for placement of positive and negative controls. The assay protocol for confirmation was essentially the same as that described in the qHTS protocol section. A Flying Reagent Dispenser (FRD, Aurora Discovery, presently Beckman-Coulter) [28] was used to dispense reagents into the assay plates.

Target-deconvolution assays

Assays were performed at 25uC in 0.1 M potassium phosphate, pH 7.4, 10 mM EDTA using 100 mM NADPH. The Prx2 assay was based on the reduction of H 2 O 2 by Prx2 in the presence of GSH measured by the reduction of the GSSG produced in a coupled assay with yeast glutathione reductase monitored by observing the decrease in A 340 nm due to consumption of NADPH (e 340 nm = 6.22 mM 21 cm 21 ) during the first three minutes [18]. The activity of TGR was determined with 3 mM 5,59 dithiobis(2nitrobenzoic acid) (DTNB, Ellman's reagent) [29] following the increase in A 410 nm due to the production of 2-nitro-5-thiobenzoic acid (e 412 nm = 13.6 mM 21 cm 21 ) [17,18].

Results

Assay Principle, Miniaturization, and Optimization

The assay was initially developed and optimized using a spectrophotometer by following the decrease in absorbance at 340 nm associated with the consumption of NADPH substrate. During these studies, the main parameters of the assay, such as buffer conditions, concentration of each enzyme and substrate, DMSO and detergent tolerance, were tested and optimized (data not shown). The optimized assay utilized TGR and Prx2 at final concentrations of 25 nM and 50 nM, respectively. The substrates' final concentrations were 200 mM NADPH, 700 mM GSH, and 500 mM H 2 O 2 . The assay was miniaturized to 1536-well format by volume reduction and appropriate adjustment of stock concentrations of enzymes and substrates to reflect the volumes being combined. For example, the assay was started by the dispense of the two enzymes at 5/3 of their final concentration to account for the well volume increasing from 3 to 5 mL, while the hydrogen peroxide substrate was delivered as 56 solution to account for its dilution (1 mL to 5 mL final volume, see Materials and Methods, Table 1). All three types of reagents (enzymes, second aliquot of NADPH, and hydrogen peroxide) were tested and were shown to be stable overnight at 4uC, a requirement for the execution of an uninterrupted fully automated screen on the Kalypsys robotic system (Figure 2). In addition, the signal being monitored was changed from absorbance to fluorescence in order to: 1) improve the signal strength, as UV-shifted absorbance assays are generally difficult to scale to a 1536-well density (due to the combination of low extinction coefficient and short path length), and 2) minimize the quenching and inner-filter effects of a multitude of compounds which absorb light in this wavelength region [23]. In a typical uninhibited reaction in 1536-well plate, the well fluorescence changed from 370 relative fluorescence units (RFU) to 180 RFU within an eight-minute window. This resulting outcome, if registered as an end-point reading, and assuming zero change in the background, would yield a signal-to-background (S:B) ratio of only approximately 2.1. Therefore the assay was further improved by modification of the type of signal collected from single end-point to multipoint kinetic read. Thus, for each plate the reaction progress was recorded for 8 minutes at the rate of one fluorescence read every 30 seconds. Such kinetic mode data acquisition not only secured a more robust assay signal but minimized the interfering effects of dust and mildly fluorescent compounds (see Discussion).

qHTS Performance

In total, 453 assay plates were screened in one uninterrupted robotic run lasting approximately 75 hours. Dispenser malfunction resulted in deteriorated signal in two plates and since the issue was noted in real time, the two 7-point libraries containing the problematic plates, plus two DMSO-only plates, were scheduled for re-screening immediately after the end of the main run. In this manner, the re-screened series were tested using the same batch of reagents as last series of the main screen. The assay performed robustly, yielding an average Z9 value of 0.76 [30]. Overall, the Z9 factor remained flat with the screen progression, with minor shifts tracking the introduction of new batches of the two enzymes ( Figure 3A). The intraplate PAT control titration was stable throughout the screen progression, resulting in average IC 50 of 1468 nM and minimum significant ratio of 4.03 ( Figure 3B) [31]. Each library compound was tested at a minimum of seven concentrations, ranging from 57 mM to 2.9 nM, and for each well, 16 time points were collected for a total of 9,562,432 data points. The screen and the preceding optimizations and validations consumed approximately 4.7 mg of TGR and 10 mg of Prx2. The overall materials cost of the screen (not including the cost of protein production) was approximately $5,200, or 0.85 cent per sample well, with approximately 80% of the costs associated with the assay microtiter plates and 15% attributed to NADPH.

Analysis of qHTS data

Unlike traditional HTS, qHTS provides concentration responses for all the compounds screened and allows determination of an AC 50 value, defined as the half-maximal activity concentration, for each compound in the primary screen. In qHTS concentration response curves are classified as belonging to one of four groups based on efficacy (response magnitude), presence of asymptotes, and goodness of fit of the curve to the data (r 2 ). For the present screen, the activity associated with each well was computed from the change in fluorescence intensity over the time-course measurement period, normalized against control wells. In addition, the y-intercept of the reaction progress plot, typically equal to fluorescence at the first time point, was stored in the database and was used to further scrutinize purported actives. Compounds which showed activity but also had elevated yintercept values were flagged as potential fluorescent artifacts.  Overview of Actives Analysis of the qHTS results revealed 39 actives characterized by full concentration-response curves and IC 50 values of better than 10 mM. After exclusion of antimony-containing compounds, as well as various mercury-and other heavy metal-containing molecules, the following series and singletons were selected for further studies after SAR analysis (Figure 4): oxadiazole 2-oxides (5 actives out of 29 analogues in collection, IC 50 potency range between 8 mM and inactive), phosphinic amides (two compounds in the collection, one inactive and one active at 37 nM), phosphoramidite (singleton active, IC 50 of 560 nM), and isoxazolone (singleton active, IC 50 of 530 nM). In addition, a weaker series, quinolinyl sulfonamides (8 actives out of 47 total analogues in collection, IC 50 potency range between 0.6 mM and inactive), was identified but noted to contain a number of both active and inactive members which were strongly fluorescent as judged by the extreme intercept values recorded during the screen. In contrast, neither the oxadiazole series nor the singleton actives exhibited any detectable autofluorescence ( Figure 5).

In addition to inhibitors, the screen yielded a number of apparent activators, that is, compounds for which the increase in concentration led to a fluorescence intensity change greater than that of the neutral control. Upon examination of the time-course plots associated with these activators it became evident that the signal enhancement originated from high starting fluorescence which decreased during the observation window and in many cases entirely obscured the assay-driven NADPH fluorescence change ( Figure 5C). While some of these compounds might be fluorescent substrates for either TGR or Prx2, which get converted to non-fluorescent products, a large number might simply be reactive towards any one or more components of the assay milieu (GSH, NADPH, and/or H 2 O 2 ). As such, their confirmation and mode of action is subject of separate study.

Series expansion and confirmation

In order to further confirm the qHTS actives and to expand the actives series, especially around the otherwise attractive singletons, powder samples were purchased from the original compound vendors and processed as described in the Methods. In addition to qHTS-identified compounds, untested analogues of the singletons were also procured in an attempt to support the singleton findings by the generation of small SAR series. The comparison of qHTS results, where applicable, and re-test results from independently acquired powder samples are shown in Figure 4, first and second data columns, respectively. The overall confirmation rate was excellent with the exception of the sulfonamide series of actives, which showed wide shifts between qHTS and confirmatory assay (results not shown). The apparent lack of confirmation for this series was consistent with the aberrant fluorescent values associated with many of its members. An analogue series built around a lowerpotency benzoindolone singleton (NCGC00038549, IC 50 of 3.9 mM) failed to yield activity against the screening assay and against both TGR and Prx2 individual assays. The original activity of that singleton was therefore deemed an artifact.

Gratifyingly, the previously-untested analogues of the phosphinic amides (compounds 1-4), phosphoramidite (13)(14)(15)(16), and isoxazolone (10)(11)(12) actives all showed activity with various degrees of potency, supporting and expanding the qHTS findings. Specifically, the ''gap'' in potency between the highly active 3 (NCGC00042730, qHTS IC 50 of 37 nM, confirmed at 25 nM on re-test) and the inactive distant analogue 4 (NCGC00064648) was filled partially by the newly-acquired analogues 1 (NCGC00093512, IC 50 of 247 nM) and 2 (NCGC00093512, IC 50 of 23 mM). Similarly, increased potency was achieved by the addition of analogues to the phosphoramidite (from a singleton IC 50 of 0.5 mM to a range of 0.2-2 mM) and the isoxazolone (from a singleton IC 50 of 0.5 mM to a range of 0.1 mM to 9 mM).

Target deconvolution

After establishing the activities of primary hits and new analogues against the screened dual-enzyme system, the compounds were further subjected to target deconvolution experiments. When tested against Prx2 in a GR-coupled hydrogen peroxide reduction assay none of the selected compounds showed activity up to the 50 mM top concentration tested (and by extension, none were active against GR, an enzyme related to TGR). Prx2 was therefore ruled out as the target of any of the actives identified in the screen. Results from the TGR assay are shown in the last column of Figure 4. The majority of active compounds demonstrated approximately the same, and in some instances improved (most notably with 7 and 8), potency against the isolated TGR as they did against the dual-enzyme system. These results not only confirm the initial findings from the screen, but also further support the hypothesis of TGR being the sole target of these actives. Additionally, all members of the sulfonamide series were inactive against both Prx2 and TGR, further strengthening the argument that their initial classification as actives was due to fluorescence interference originating from either the compound, impurities, or product(s) of its breakdown.

Discussion

The stability, relatively low cost, and effectiveness of praziquantel has practically created a dependency on this single drug to treat schistosomiasis. Both the success of praziquantel and the general lack of incentives for large pharmaceutical companies to embark on research and development in the area of tropical diseases have led to a fairly dry pipeline for both drugs to treat schistosomiasis and basic research tools to study the lifecycle of this important parasite. To this end, we implemented a highlyminiaturized automated screen of the NCGC small molecule collection in an attempt to identify novel inhibitors of S. mansoni TGR or Prx2, both of which have been recently validated as crucial S. mansoni enzymes and have been proposed as targets for drug development. Prior to HTS adoption, the assay employed monitoring NADPH absorbance. While such a format is very convenient, offering fast access to kinetic data via the use of standard spectrophotometers, measuring absorbance in the UV region in 1536-well density is rarely practical. A significant fraction of organic molecules, as well as dust and buffer components, absorb in the 350 nm range, thereby introducing unacceptably high levels of interference. Additionally, the relatively low extinction coefficient of NADPH coupled with the short optical path length of the plate well significantly reduces the signal available for detection. Because NADPH is naturally fluorescent, emitting at ,450 nm, while its oxidized counterpart NADP is not, we switched the detection platform for the coupled reaction from absorbance to fluorescence, a step that parallels the application of profluorescent substrates in assays for phosphatases and proteases [23], with the main difference being the fluorescence change trending from high to low in this reaction.

Because of the anticipated fluorescence interference from compound library members in this blue-shifted detection region and because the output generated from NADPH is not very strong (due to the combination of low extinction coefficient and quantum yield), we further modified the detection format of the assay to measure the reaction progress in kinetic mode as opposed to collecting a single end-point read. While kinetic, or time-course, measurements are routinely performed during assay development in low-throughput settings, their practical implementation during automated large-collection screens is not trivial. Unless the reaction under study is slow, only a fast-scanning reader or whole-plate imager (such as the ViewLux) can allow positionallyunbiased and rapid repeated measurements of 1536-well plates without significantly slowing down the overall plate processing speed. The collection of at least a two-point time course allows the effects of dust and fluorescent but otherwise inert library members to be subtracted out to reveal the true reaction course. Because the first time-point values (when the enzymatic reaction has produced minimal amount of product) associated with each compound well are stored in the database, a further analysis can be performed to flag interfering fluorescent library members [32]. An added benefit is that the signal-to-background computed from kinetic measurements significantly improves relative to end-point data and thus allows screening under conditions of low substrate conversion [33]. While in this screen we collected a total of 16 points per well, further optimization of the assay conditions could have resulted in shortened read time without the loss of sensitivity.

The primary screen against the TGR/Prx2 cascade was performed in Quantitative High Throughput Screening (qHTS) format. In qHTS, every compound in the collection is tested over a range of concentrations, spanning from tens of micromolar to low nanomolar, to generate a complete concentration-response profile. As such, qHTS is best described as high throughput pharmacology, since as a result of its application, not only are potencies and efficacies assigned to each active compound but also false positives and negatives due to outliers associated with individual concentration responses are easily identified in the context of titration. Additionally, due to the built-in replicates in the testing of each compound, the need for laborious and infrastructure-intensive cherry-picking, original-result replication, and dose-response characterization are eliminated. In our present assay, the application of qHTS enabled us to not only skip the direct confirmation steps but also to combine the actives verification from independently-sourced powders with series expansion around limited SAR or singletons. In traditional singleconcentration screening, singleton actives are necessarily treated with great caution given statistical uncertainties. In this study, the qHTS paradigm allowed us to confidently select the potent phosphinic amide, isoxazolone and phosphoramidite singletons 3, 10 and 14 ( Figure 4) for further testing and that selection was later validated by the excellent confirmation of those actives and the successful expansion of the series. Separate, but equally important, is the aspect of reliability and robustness of screening data. qHTS, with the combination of dose-survey and replicate points, indeed offers uniquely rich and robust data sets for deposition in recently established public databases, such as PubChem. Additionally, in order to minimize the interfering effect of promiscuous inhibitors acting via colloidal aggregate formation [34,35], we included detergent in the assay buffer.

Throughout the entire screen, the assay performed in a robust manner, yielding an average Z9 value of 0.76. Overall, the Z9 factor remained flat with the screen progression, with minor shifts tracking the introduction of fresh batches of enzyme. The availability of periodically computed Z9, signal-to-background, and activity heatmaps throughout the screen progression, made possible by the development of fast data-processing tools in-house, significantly improved our response time when screen complications arose. For example, the dip in Z9, also accompanied by noisy activity heatmap (not shown), was noted almost in real-time and this allowed the appropriate concentration series to be scheduled for re-run within the same screening session. Figure 3B presents a cumulative plot of all intra-plate concentration-response curves of PAT throughout the entire screen. The narrow range of observed IC 50 values serves as a further indication that the screen performed robustly from a standpoint of enzyme activity and responsiveness to inhibition. The titration curve displayed stability throughout the screen despite the fact that PAT is only partially soluble in DMSO and required formulation in highpercent water, leading to concerns about evaporation-related variability. Analyzing the trend in the intra-plate control as a function of screen progression allows one to ascertain the 'health' of the screening system as a whole, because the variations or dramatic shifts in potency of the control could be due to not only a deterioration in enzyme quality (which could otherwise be detected from an shift in the S:B value) but also to problems with the pintool delivery of compounds. The absence of abrupt and significant shifts in the intra-plate control curve allows us therefore to conclude that the compound transfer remained uniform throughout the screen.

The screen identified numerous arsenic, antimony, mercury, and other heavy-metal containing compounds (data not shown; for complete set of actives, see PubChem, AID 448). The antimonycontaining compounds were largely similar to PAT and were therefore expected to be identified by this assay. Likewise, Hgderivatives inhibited the enzymes strongly, as expected. While PAT and the gold-containing drug auranofin had been shown to inhibit thioredoxin reductase and TGR [17,36,37], and while more recently arsenic trioxide has shown anticancer activity and its effect has been ascribed to Trx reductase-mediated apoptosis [38], we restricted our analysis to novel nonmetal-containing compounds, primarily due to the fact that heavy metal-containing compounds frequently exhibit non-selective inhibition against a broad panel of enzymes and because nonmetal novel chemotypes against these previouslyunscreened targets appeared to offer the greatest promise for optimizing potency and specificity. In this regard, the screen was successful, having resulted in identification of several distinct series of TGR inhibitors. It is noteworthy that the top actives from some series, such as the phosphinic amide 3, oxadiazole 2-oxide 7, and isoxazolone 11, yielded IC 50 values close to the final TGR assay concentration (25 nM in qHTS and confirmation and 15 nM in the TGR individual assay), thus approaching the limit of the detectable potency range. In terms of the lead actives there are several interesting points. For instance, the role of the benzothiazole heterocycle within the phosphinic amide series is apparently critical for inhibition as illustrated by the comparative values of analogues 3, active and containing a benzothiazole moiety, and 4, inactive and devoid of benzothiazole ( Figure 4). The oxadiazole series contains several symmetric heterocyles (a function of their synthetic ease) and are known NO donors [39,40]. The presence of two phosphorus based small molecules may well relate to the presence of a selenocysteine in TGR and the relative electrophilic nature of this functionality. Studies to expand upon selected lead actives and further understand their mechanism of action are currently underway. Furthermore, in the Prx2 deconvolution assay, none of the top actives were found to inhibit GR, an enzyme closely related to TGR. This suggests that selective activity against the parasite (which lacks GR) and less toxicity to humans (who have GR) can be achieved.

In summary, a kinetic-based qHTS against a pair of novel, validated targets from S. mansoni allowed fast and reliable identification of compounds active against this critical redox cascade. We have identified several novel structural series of TGR inhibitors, several of which are highly potent and should serve both as mechanistic tools for probing the redox balance in S. mansoni, and starting points for developing medicinal leads for much-needed new treatments for schistosomiasis. The work presented here effectively bridged the gap between academic target identification and the first steps of drug development for an important neglected disease [20]. Generalization of this paradigm to other neglected diseases could prove be a powerful approach to catalyzing new therapeutic development for NTDs.

Figure 1 .
1TGR/Prx2 assay principle. doi:10.1371/journal.pntd.0000127.g001

Figure 3 .
3qHTS performance. Z9 trend (A) and intraplate control titrations (duplicate curves per plate) (B) as a function of plate number. doi:10.1371/journal.pntd.0000127.g003

Figure 2 .
2Screening reagents' stability as a function of storage time. Bottles containing buffer, enzyme, and substrate stock solutions, respectively, were prepared and kept at 4uC. At the selected time points, the bottles were connected to a liquid dispenser and the assay was performed as described in Methods. Signal-to-background ratio (S:B) and Z9 factor were computed as the average of 32 wells. doi:10.1371/journal.pntd.0000127.g002

Figure 4 .
4Structure and potency (IC 50 in mM) of actives and series expansion analogues. 'qHTS' lists the IC 50 values obtained from the initial high throughput screen of the collection; 'confirmation' represents the IC 50 values obtained using the identical assay as in the qHTS from the compounds identified in qHTS and expansion series; 'TGR Assay' is the IC 50 values obtained in an assay of TGR activity using DTNB as a substrate. NS, not screened (compound not in the screening collection), ND, not determined, NA, not applicable. IC 50 values greater than 50 or 57 mM signify lack of fitted curve through the dose-response data, i.e. flat response within the range tested. doi:10.1371/journal.pntd.0000127.g004

Figure 5 .
5Example time-course plots derived from the primary screen, followed to the right by the corresponding concentrationresponse curves. (A, B) Non-fluorescent inhibitors, compounds 3 and 14; the decrease in fluorescence is inhibited at increasing compound concentrations while the starting fluorescence remains constant. (C) An example of an apparent activator (Ofloxacin): a highly fluorescent compound which appears to be reacting with one or more components of the assay. Four concentrations out of seven or fifteen are plotted in each of the time course plots for clarity; &, 3.2 nM; m, 16 nM; %, 10 mM; n, 50 mM. doi:10.1371/journal.pntd.0000127.g005

Table 1 .
1S. mansoni redox cascade qHTS protocol.Step
Parameter
Value
Description

1
Reagent
3 mL
Enzyme and no-enzyme control solutions

2
Library Compounds
23 nL
57 mM to 2.9 nM titration series

3
Controls
23 nL
PAT titration

4
Incubation Time
15 min
Compound interaction with targets

5
Reagent
1 mL
Second equivalent of NADPH

6
Reagent
1 mL
H 2 O 2 Substrate addition to initiate reaction

7
Assay Readout
365/450 nm
Fluorescence intensity kinetic read

Step Notes

1
Black solid bottom plates, single-tip dispense, enzyme in columns 1, 2, 5-48, no-enzyme in columns 3 and 4.

2
Pintool transfer of library samples to columns 5-48 of the assay plate.

3
Pintool transfer of PAT titration to lower half of column 2.

4
Room temperature incubation in auxiliary hotel.

5
Solution of 400 mM NADPH and 700 mM GSH.

6
Hydrogen peroxide addition via 8-tip dispense.

7
ViewLux CCD imager, one read per 30 seconds for 8 min.

doi:10.1371/journal.pntd.0000127.t001

PLoS Neglected Tropical Diseases | www.plosntds.org
PLoS Neglected Tropical Diseases | www.plosntds.org 7 2008 | Volume 2 | Issue 1 | e127
AcknowledgmentsWe acknowledge S. Michael and C. Klumpp for assistance with the automated screening, Esther Dubrovsky for technical assistance, and Angela N. Kuntz for assistance with enzyme preparation. The quantitative high-throughput screening dataset has been deposited in PubChem under Assay ID 448.Author Contributions
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A teacher's identity trajectory within a context of change

Marietjie Oswald email@example.com
Department of Educational Psychology
Faculty of Education
Stellenbosch University
South Africa

Mariechen Perold
Department of Educational Psychology
Faculty of Education
Stellenbosch University
South Africa

A teacher's identity trajectory within a context of change
5DFF4629C4F9F6841ED5912A2FD39AE9agencyCHATdisadvantaged contextemerging economyidentity trajectorypractical activityprimary schoolteachertransformation
This article examines the effects of political, socio-economic and educational change on a South African teacher's identity trajectory.Our research was conducted at a primary school in a historically disadvantaged community in the Western Cape Province, South Africa.We applied a cultural-historical activity theoretical (CHAT) lens to explore the identity trajectory of one of the teachers.Our findings suggest that a teacher's identity is a social product, drawn from social history, actively internalized and re-authored in response to new circumstances.This was especially evident during the transformation at the macro-political, educational and institutional levels in South Africa since 1994.

Introduction

In this article, we report on an attempt to make sense of the effects of political, socio-economic and educational change in South Africa on a teacher's identity trajectory.The data was generated during a university-school research partnership that focused on teachers' situated practices of care and support.The majority of the participants had worked as teachers for an extended period, and lived through most of the many decisive changes in South Africa over the last few decades.The findings showed that the narratives of their care and support practices were intertwined with their general experiences as teachers.Their stories highlighted the different ways they made sense of the multitude of changes in the political and social spheres, and how these affected their lives and career trajectories.

In particular, we examined the effects of such changes on the identity of one of these teachers, whom we will call Iris, to ensure her anonymity.Iris was articulate, a strong voice in the school, eager to share her experiences and willing to collaborate with us in making sense of her career as a teacher during a time of change.

For our theoretical framework, we chose CHAT.When taking a CHAT approach, the researcher needs to take into account the impact of history's large footprint on a subject's private and public life.A person can only be understood in terms of his or her own context and history.Gutiérrez (2002:319) claims that "the strategic use of social categories can be productive".In order to make meaning of Iris's identity trajectory we needed to return to those social categories legislated during the apartheid regime.

Iris is a member of the so-called 'coloured' population in South Africa.This group had its origins in the intermixing between slave groups, indigenous groups, and white settlers in the Western Cape from the 1650s onwards.By imposing the label 'coloured' on this group, the all-white Nationalist government deprived them of political status -'coloureds' were officially neither black nor white.The 'coloured' community was thus subordinated to the dominant white community and excluded from many socio-economic, political and educational privileges (Sonn & Fischer, 1996).

The race-based inequalities that were created resulted in the population being fragmented along racial lines.Certain communities -despite, or perhaps even because of segregation -found innovative ways and alternative activity systems through which they survived and maintained their cultures and identities.Sonn and Fischer (1998) claim that literature and research on group responses to oppressive systems tend to emphasise negative outcomes, and the victim status of these groups, without considering the element of community resilience.They argue that community membership is an essential source of wellbeing, can act as an antidote to oppression, exclusion and psychological hardship, and can be integral to the survival of valued cultures and identities.It can also offer a sense of historical continuity.

Apartheid acted as "a powerful allocator of identity" and frequently "suppressed identity through centralizing [sic] race and ethnicity at the expense of other markers of identity" (Singh, 1997:120).Its abolition brought South Africans the opportunity to transcend and reshape new identities, to fashion new understandings of who they were and what they considered fundamental to themselves (Singh, 1997).Thus, dramatic sociocultural changes have occurred in South Africa since 1994 (Moloi & Henning, 2006).Since the inauguration of the new democratic dispensation and the newfound freedom, all the race groups have needed to reconcile themselves to a redefinition of their previously-held identities.In many cases, they have also needed to reconsider their positioning in society.This has resulted in the resurgence of issues of identity (Francis & Le Roux, 2011).Despite the initial elation with which the historically marginalised groups welcomed their newfound freedoms, the radical nature and extent of the transformation nevertheless caused considerable stress for them and everybody else in South Africa (Van Niekerk & Prins, 2009).

The race-based inequalities in South Africa reverberated in the economic sector, and overlapped with the change to democracy.While poverty was virtually non-existent among whites, at least 58% of all South Africans in general -and 68% of the black population in particular -were living in poverty in 1995.These inequalities pervaded education, health and basic infrastructure (Ozler & Hoogeveen, 2005).Currently, South Africa is recognised as an emerging economy, characterised by a rapid pace of economic development and a substantial effort to liberalise the economy (Meyer, Estrin, Bhaumik & Peng, 2009).Despite substantial progress since 1994, several critical challenges are still impeding the achievement of South Africa's considerable potential (Organisation for Economic Co-operation and Development (OECD), 2013).In 2012, the Gini coefficient for South Africa was about 0.60 (Landman, Bhorat, Van der Berg & Van Aardt, 2003), one of the most unequal income distributions in the world.Today it is estimated that about 45% of the population still live in poverty (Landman et al., 2003), with almost all of the poor belonging to the black or coloured groups ( Van der Berg, Burger, Burger, Louw & Yu, 2006).South Africa's unemployment rate is one of the highest in the world, while poverty, especially extreme poverty, has not been alleviated (Ozler & Hoogeveen, 2005).

The OECD Economic Survey of South Africa (2013) identified education as another of the country's key problems.The report claims that the poor quality of education in South Africa acts as a serious impediment to economic growth.A distinguishing feature of the education system remains the very high degree of inequality between schools.This legacy of apartheid is one that the present government is finding hard to address.Although there is one overarching Department of Basic Education for all schools, there are still massive inequalities between the formerly advantaged schools (for white learners) and formerly disadvantaged schools (for the rest).This is especially evident in many rural areas -including the community in which Iris taught -where poverty in all its manifestations is the principal feature (Engelbrecht, 2006;Taylor, 2007).According to Bloch (2009:25), "education as it stands today continues to reproduce inequalities in society, inequalities that threaten the stability and comforts of young people."

A report issued by Wits Education Policy Unit (2005) claims that before 1994, South African teachers were repressed, with very limited professional autonomy.The education of teachers was underpinned by a conservative pedagogy, which instilled passivity and obedience to authority.Critical or independent thinking were not valued.Their work was strictly controlled through a system of inspection, which undermined their autonomy.They came to rely on the agency of the state to direct them in doing their work.A loss of selfconfidence and self-reliance resulted.

Teaching philosophies have changed radically since 1994.Approaches and practices are now based on democratic, inclusive and participative relationships, reflective practice, experimentation and risk-taking.Teachers have been called on to learn new theory and to implement new practices in their classrooms (Oswald, 2010).Moreover, in an effort to accelerate the transformation process, they have been subjected to a flurry of policy initiatives in a top-down fashion.They have had to attend numerous workshops and been subjected to extreme answerability and accountability measures.In the years since 1994, they have gradually lost confidence in the education authorities and have begun to blame them for the many challenges they are forced to face in schools and classrooms, without the necessary assistance and support (Perold, Oswald & Swart, 2012).

To compound matters, the teachers themselves have received negative media publicity and are frequently blamed for the failure of educational innovations, reflected in the underperformance of South African learners (Perold et al., 2012).Comparative international studies, such as the Trends in Mathematical and Science Study (TIMSS), the Progress in International Reading Literacy Study (PIRLS) and national criteria such as the Department of Basic Education's Annual National Assessments (ANA), confirm that South African learners underperform.This is particularly evident in schools in vulnerable communities, such as the farm school where Iris taught.Despite the negativity surrounding education and teachers, in particular, the strategic importance of teachers in the South African education system cannot be denied.In a time of change, they are expected to act as agents of change and have a key role in ensuring that quality education is delivered to all children.

The above forms the backdrop to the question that directed our research: How does Iris's identity trajectory illuminate the impact of apartheid on the self of a person less privileged by a particular social and political regime, as well as the complex changes that have occurred on macro-social and educational levels in South Africa since the inauguration of the new democratic dispensation in 1994?

In the next sections, we present our chosen theoretical framework and Iris's identity trajectory as a teacher.We conclude with a brief discussion.

Cultural-Historical Activity Theory (CHAT) as Framework for the Study

CHAT departs from essentialist or context-independent notions of identity, instead viewing the self as embedded and intrinsically interwoven with socio-cultural and historical processes.Researchers in CHAT accept the challenge of "rendering an account of the self as a profoundly social phenomenon, yet at the same time as real, agentive and unique" (Stetsenko & Arievitch, 2004:476).

Vygotsky's work is fundamental to all contemporary variants of CHAT, but was further refined in the seminal work of Leontiev.Stetsenko and Arievitch (2004) and Stetsenko (2005) articulate human subjectivity (as derived from the work of Leontiev) as originating from and existing within activity processes.This eliminates the dichotomy between individuals and the world.Practical, social and purposeful (object-related) activity is the principal form of human life (Stetsenko, 2005).In CHAT, therefore, analysis of the agency of human subjectivity and inter-subjectivity needs to keep in view their ultimate origin in material processes of human activity.

Stetsenko and Arievitch (2004:476) expand Leontiev's work on object-related human activity by conceiving of CHAT as having an "ontological unity of inter-individual and intra-individual processes", as accounted for in "transitions between the self and broader sociocultural processes".This implies that an individual, such as Iris in this study, is simultaneously formed by society and informs society; that she is created by history but that she also creates her own history, and that she is the "author and actor of her own drama" (Marx, 1955, in Stetsenko, 2005:85).

These possibilities for agency make identity and identity processes important in the working life of a teacher such as Iris.In the words of Holland and Lachicotte (2007:134-135), Iris is "inhabitated by [the world], and yet is able to co-construct the social and cultural world" in which she exists.Phrased differently using the metaphor of residence, this means that although Iris inhabits the social structures and spaces that have been created by the collective society, she nevertheless can produce a self which inhabits these structures and constructs in creative and even subversive ways.This particular argument is significant in the context of this article, and will be extended in our discussion of Iris's identity work.

Method

Our active engagement at the research school spanned a period of three years from 2008 to 2010.We employed an ethnographic design for the project.The principal and the staff were approached, and once they had indicated their willingness to take part in the research, ethical clearance was obtained from the Research Ethics Committee at the relevant university.The project was further approved by the Western Cape Education Department (WCED) before being launched at the school.This primary school is situated in a rural area in the Western Cape, where the majority still suffer harsh conditions of poverty, adult illiteracy, social breakdown and disease.Despair, a sense of hopelessness and a fatalistic worldview are frequently the result.At the time of our engagement with the school, it was small, with 12 teachers and 380 learners.The teachers were not members of the community the school served, but travelled daily from more affluent areas, thereby creating a socio-economic and geographical distance between themselves and the school community.Besides their teaching roles, they were often the main source of care in the immediate environment (Perold et al., 2012).Like teachers elsewhere in historically disadvantaged schools, those in Iris's school struggled to address the manifold and diverse needs of their learners, especially given the lack of resources.

We employed a qualitative methodology and data was generated through three group discussions with the staff, two focus group interviews with four teachers, an individual interview with Iris (pseudonym) and open-ended questionnaires completed by all the teachers.A narrative style of respectful curiosity (Madigan, 2011) was utilised in interactions with all the research participants throughout the gathering of data, aiming to elicit their knowledge and experience of their teaching and support practices.The conversations we had with all the participants and Iris were aimed at providing them with opportunities "to generate identity conclusions informed by non-structuralist categories of identity -intentions and purposes, values and beliefs, hopes, dreams and visions, commitments to ways of living, and so on" (White, pers. comm., 1992, in Madigan, 2011).To answer our specific research question, we set out to capture Iris's experiences of her career trajectory, since she began working in education.For some time, she had kept a diary to help her to make meaning of her experiences.She agreed to share relevant extracts from this in a life-line format.The data obtained from the life-line exercise was analysed and served to inform further questions which we posed in a further individual interview with her.We revisited our first data set, obtained as part of the original project, as secondary data, adding richness to the more recently generated primary data.

CHAT usually views data analysis as an abductive process, which implies a movement between an inductive and deductive process of knowledge creation (Foley, 2002;Rautkorpi, n.d.).During the first phase of data analysis, we approached the primary and secondary data from an inductive stance, taking a tentative position.Global analysis, a tool used "for thematic, networked analyses" (Henning, Van Rensburg & Smit, 2004:109-114), was considered appropriate for interpreting our data sources.Such analysis can include a whole range of analytic procedures sharing the characteristic that data is read for broad patterns and themes, which can be meaningfully linked into an integrated whole (Henning et al., 2004).

The end-product of the first phase of data analysis revealed broad themes and patterns in Iris's experiences, both as a teacher and as a person.The deductive facet of the abductive process was invoked by positioning the themes and patterns derived from the raw data within the more recent literature in the CHAT tradition.As the data was generated in Afrikaans, Iris's home language, we translated the direct quotes reported in the article into English after the analysis.

The following ethical considerations, as suggested by Babbie andMouton (2001), Henning et al. (2004) and Terre Blanche, Durrheim and Painter (2006), were upheld during the research study and in particular in our engagement with Iris: care was taken to acknowledge her privacy and to address her with sensitivity; her right to anonymity, confidentiality and voluntary participation was respected, and our interviews with her and other research activities in which she took part were conducted within a relationship of collaboration, trust and transparency after she signed a consent form.

The next section explores Iris's identity trajectory as embedded in CHAT, against the backdrop of the many changes that have taken place in South Africa, especially since 1994.

Iris's Identity Trajectory as a Teacher

The following description of Iris's identity trajectory as a teacher is based on the themes identified in the different data sets, presented chronologically and superimposed on CHAT.As we have noted, this theory views identity not as static and coherent, but as variable, multi-vocal, interactive and mutable across contexts and over time (Holland, Lachicotte, Skinner & Cain, 1998, in Daniels, 2007).

The dramatic rupture in the political dispensation in South Africa since 1994 and the possibilities that this has generated for change in all walks of life has made the relearning of identity inevitable (Geijsel & Meijers, 2005;Singh, 1997).Historical factors and changes in the socio-cultural domain play a pivotal role in Iris's work as a teacher and straddle her public and private lives.We traced her identity trajectory in a narrative format, following themes which specifically emerged from the data: her childhood experiences up to the time of leaving school, her years of studying to become a teacher, the early years of her teaching career, the 29 years (as at 2013) at her current school, and her role as a mother.

Iris was her parents' eldest child.Her earliest memories are of how her family initially lived in servants' quarters, moved temporarily to the centre of the town where she grew up, and were forcibly moved to a rented house in an area designated for the 'coloured group' on the outskirts of the town.Through the practiced segregation from the white community, given higher social standing, they experienced instability and felt displaced.

Although these forced removals had a detrimental effect on the identity of this particular 'coloured' community, a strong and cohesive alternative community eventually developed (Sonn & Fisher, 1998).This occurred at two levels: the externally constructed, imposed and negative label of the community as 'coloured', and an internal construction mediating activity systems, which imbued its members with "security, stability, belongingness and psychological relatedness" (Sonn & Fischer, 1998:466).Throughout Iris's life she was nurtured by strong communal support reminiscent of Sonn and Fischer's (1998) second level community.

Despite considerable financial hardship, Iris's father managed to procure a plot in the designated area, where he built a house which gave his family a standing in the community.He also inspired the young Iris to study hard and to equip herself with an education that could lend her some agency in a country ruled by the apartheid ideology.Iris failed her grade during her first year in the new school, as she relates: "in the first year many, many learners failed.I also failed."She further related that her experience of 'not making the grade' was directly linked to her family's displacement and forced relocation to a new area.This highlights the interrelatedness of the self and the social: the social inhabits the self, which manifests in the individual's actions and experiences (Stetsenko, 2005).Iris's position as a child living in a 'coloured' community before 1994 made her particularly vulnerable to forces outside her control, affecting her actions and agentive possibilities.

Iris's father died suddenly when she was 13.He was a strong influence in her life and the unexpected tragedy brought new changes and challenges.Her mother, a strong woman and an exemplary role model for Iris, took over and helped Iris to counteract the negative effects of their circumstances.Iris learned to adapt and to make alternate plans.She left school after Grade 10.This was contrary to her father's dreams for her, but was unavoidable given the family's financial constraints.However, she managed to enrol in a teacher training college in a nearby town.She flourished in her new-found circumstances, noting of her experience: "this was one of the best times in my life.This period in my life really enriched me."She became independent and this paved the way for her younger sisters to follow in her footsteps, since they too became teachers.

Her identity was now established, both as the leader in her family and as a professional teacher.This period in her life coincided with the unrest following the 1976 Soweto uprising.During the subsequent years of activism, Iris became more politically aware.She gained a broader understanding of how activities and decisions on a macrolevel had invaded her subjectivity and that of her apartheid racial designation.For the first time, she could identify with her mother's sadness when forced to sell some treasured family furniture, only to see it offered for sale later at an antique shop in town for much more than they were originally offered.

Iris began to question the dominant discourses in the country, and their negative impact on her identity.At the same time, she found solace in her community; there she felt valued.She articulated her response to this in the following way: "but I learned not to be bitter about it, to put the past behind me, stay focused and to move forward."She also met her future husband while studying and the role of 'cherished wife' became part of her identity.After completing her basic training as a teacher, she went on to specialise in class music.This contributed significantly to her identity as a teacher: I [would] not change anything about the experience that I gained while studying.I learned about discipline, being on time, and responsibility.I shall always be grateful to my mother for making this possible.

Iris procured a position as a Grade Three teacher in a primary school in the town where she had received her training, and started her teaching career in 1978.She loved her work as class music teacher, and was able to use all the expertise gained during her training.She remembered the principal of this school as an important mentor, from whom she learned valuable lessons.She described him as "being like a father to [her]".Teaching as an activity was central in her life and informed her identity.She flourished in the school and valued the collaborative collegial relationships she formed during her time there.

She worked in a structured environment with clear guidelines.Her initial training as teacher was seen as adequate for her position as classroom teacher, her knowledge and skills were valued, and she was not expected to gain any additional skills by attending workshops (as she would from 1994 onwards).Her pupils could learn, read and do mathematics.Routines were predictable, there were no surprises, no failures and "the 'department' provided everything I needed, from the curriculum to the necessary resources."This echoes the statement from the Wits Education Policy Unit (2005) that during the apartheid dispensation, teachers were both controlled by and reliant on the state.

This observation speaks to one of the paradoxes in Iris's identity trajectory.From a historical perspective, it could be argued that teachers were robbed of any form of agency by both the rigid state control of education and by their own lack of critical awareness.Nevertheless, the more structured environment in which Iris taught, as well as the sound progress of her learners, gave her a sense of safety and a feeling of accomplishment.State control was evident in the inspector system that was employed to appraise performance.Despite this, Iris recalls having only a minimum of administrative record-keeping responsibilities -in sharp contrast to the current demands on teachers.She interpreted this minimal state intervention as an affirmation of trust in her abilities and a confirmation that her work was good.She defined her role as a teacher as being "responsible for teaching and learning".Iris particularly cherished this part of her career.Her goal-directed activity as a teacher, aimed at the successful progress of her learners, delivered positive outcomes.Stetsenko ( 2005) emphasises goal-directed activity as the driver for the development of human subjectivity in the reciprocal influence between the individual and the social.Thus, in this period, two factors impacted on the development of Iris's identity trajectory as a teacher in this culturally constructed and socially imposed world (Holland & Lachicotte, 2007).Firstly, the safety of a structured environment, which allowed her to feel that she was in control and an expert at what she did, and, secondly, her pride in being a provider to her family.

Iris joined the staff at her current school in 1984.As previously noted, this was a farm school in a vulnerable community in the Western Cape.The teachers were confronted by severe challenges due to the adverse circumstances which their learners faced at home and in their community (Perold et al., 2012).Contrary to Iris's experience at the previous school, the first two principals at this school both employed autocratic management styles, ruling with an iron fist.Nevertheless, she still enjoyed teaching.She also furthered her own education, achieving her Grade 12 qualification through distance learning.She subsequently registered for a Bachelor of Arts (BA) degree at a university for 'non-white' learners, and successfully completed the degree.She mentioned in her diary that apartheid had made it impossible for her to attend a nearby so-called 'white' university.However, she made full use of available opportunities to further her education, despite the limiting conditions afforded her by society.This more goal-directed activity contributed to her viewing herself in a positive light.

Iris talked with pride about her many accomplishments with school choirs.These had won prizes at local competitions; even colleagues with more years of experience acknowledged her expertise in the field.During these years, she and her husband bought her mother's house (the one her father had built many years before), renovated and extended it.She also "went to a big academic hospital for infertility treatment in order to start with a family".She established a stable and successful middle-class family, despite the broader social, political and economic disruptions that were racking the country in the dying years of apartheid.

Nineteen ninety-four was a watershed year in South Africa's history.The new democratically elected government abolished discrimination on the basis of race and feelings of euphoria and optimism abounded.However, the radical nature and extent of transformation caused stress for many teachers, despite their initial elation at their newfound freedoms (Van Niekerk & Prins, 2009).Iris and her colleagues, as she put it, "had to make huge paradigm shifts" in being retrained for the changed educational dispensation.They were obliged to attend numerous workshops and felt unsure of what was expected of them.Even the language changed: they were now called 'educators' instead of teachers, while pupils became 'learners'.As if this were not enough, they were inundated with files with information on new ways of teaching.Iris described how she became an "educator as well as student" Tasks doubled, administrative workload expanded, and I found myself not having enough time to teach the way I was used to and what I believed in.Many teachers left education and were not replaced, thus classes became bigger and bigger, and I became a 'crowd-controller' instead of a teacher.

The problem of adapting to curriculum changes has become a hallmark of teaching in the 'new' South Africa.Since 1995, teachers have had to adapt to three curriculum changes.The highly criticised Outcomes-Based Education (OBE) system was introduced in 1998 and modified in 2007.It is currently being replaced by the National Curriculum Statement Grades R-12, based on a different set of philosophical underpinnings.Iris complained that the revised practices following from these curriculum changes have not been conducive to learning.These factors -particularly the way in which new language has shaped new roles and new descriptions of the work expected of her -led to confusion and despondency in her working life.She confessed to feeling like she was running around like a "headless chicken [sic], keeping extremely busy with frenzied activities in order to keep my head above water".Her activities had become less effective, the expectations of the education authorities confuse her, and, most significant of all, the "children cannot read, write or do maths!"Her identity as a strict but competent teacher has been eroded by a feeling of failure.She felt as if "she [was] caught up in a wrong world.I have never before felt so stupid, dull and confused![sic] Just plodding along, just plodding along every day!"

Iris seemed to have lost a sense of personal agency.She reports that blaming others remained her only defence against the fact that learners were not making progress and that teachers were seen as not making the grade by both the education authorities and the media in South Africa (Bloch, 2009;Perold et al., 2012).She blamed learners for not taking responsibility for their own learning, parents for lacking parental skills ("parents do not address gnats, they wait for the elephants" [sic]), outsiders becoming involved as do-gooders in the school, and then using up precious time that could have been spent on teaching (maybe trying to ease consciences), and excessive departmental expectations ("I have to be social worker, pastor, doctor, nurse, psychologist and more").Iris's cry for help could well resonate with many teachers in South Africa: "I am tired, depressed, despondent and fedup [sic].I see a very dark future for education in South Africa.When will I be allowed to teach again?"

Iris felt both that she was a failure and that she was being "crucified [sic] for being a failure".She felt she had to "pick up the pieces" of her confidence, but did not know how.Her agency as teacher was being diminished and the demands on her were making inroads on her confidence.Her work as a teacher was negatively impacted by these social-cultural demands, which in turn shaped her identity, and recursively affected her teaching practice and how she viewed the current educational dispensation.Her reaction to her situation nevertheless revealed a further paradox in her identity trajectory.Despite painting a bleak picture of education in South Africa and seriously questioning her own future as a teacher, she was not prepared to give up: "Me throwing in the towel?Never! [sic]" Several factors contributed to her determination to carry on, regardless of the overwhelming challenges facing her.First, 18 years ago, she and her husband adopted a baby girl.Motherhood has given her the opportunity and space to live out her original ideals about education.She supported her daughter in all her academic and extra-mural endeavours and, through her success as a mother-who-is-also-a-teacher, confirmed her own identity as a teacher.Second, as a result of her experiences during apartheid, Iris was adept at improvising across the limits of cultural traditions and social forces of power and domination to find spaces in which to re-invent the self (Holland & Lachicotte, 1998, in Daniels, 2007).In this way, she found the determination to carry on, regardless of her current despondency.

Conclusion

Iris's identity trajectory was loaded with complexities, ironies and paradoxes.CHAT helped us to make meaning of the data, offering us a robust and broad-based platform from which to engage with her identity trajectory (Oswald & Perold, 2011).We came to understand how this had unfolded, as the outcome of the various activities in which she had participated, both as a teacher and as a human being, during divergent political periods in South Africa (Stetsenko, 2005).These activities provided the background for the unfolding of her identity trajectory as changes in object-related activities seemed to contribute to Iris's understanding of her identity.It was also clear how her identity derived from an "ensemble of societal relations" (Marx & Engels, 1968, in Roth, 2007:84).This was evident in the narrative of her life, from her birth in the 1950s in a segregated country in which she was marginalised on grounds of her race, to her positive experiences -despite considerable political constraints -as a young teacher, to her current sense of disillusion over the perceived failure of the education authorities to support teachers and to ensure quality education for all learners.Our study offers proof of CHAT's conceptualisation of the self-in-development as located not "under the skull" but in the processes of ongoing practical activity and social transactions (Stetsenko & Arievitch, 2004:475).

A modest form of agency was evident in how Iris was "inhabitated by [the world]" but was still able to "co-construct the social and cultural world" in which she exists (Holland & Lachicotte, 2007:134-135).Her social position until 1994 was shaped by a history which allowed her as a member of the coloured community only a limited space for agency.Nevertheless, as an efficient teacher and as a valued member of a particular community, she still managed to position herself favourably, despite political and other restrictions.Today, however, her experiences as a teacher in a transformed South Africa, threaten the sense of agency that she experienced previously, and therefore also how she currently views herself.In the past, she assumed that if she did her best as a teacher, her learners would prosper.During the previous dispensation she was able to focus on this aim and her learners flourished.Currently, too many complex factors out of her control militate against this.Her learners are mostly not 'making the grade', leaving her cynical, feeling a failure and often depressed.

From all the above it is evident that Iris's sense of agency fluctuated as it was influenced by contextual changes.Regardless, she still remains committed to carrying on, although simultaneously believing that a whole generation of children, subjected to the turmoil of a transforming education since 1994, is lost.This seeming paradox in Iris's thinking illustrates the fluidity of her identity, dependent on practical activities and social transactions present in her life.

In retracing Iris's identity trajectory, we determined what a teacher might need in order to feel cared for, supported and valued.Iris referred to the importance of positive school leadership.A school leader with the capacity to act as a positive role-model for teachers can inspire feelings of value and safety, empowering them to take risks and to strive to become the best they can be.She also stressed the benefits of a school as a community, where collegial relationships could act as networks of support for all teachers, but especially for novice teachers.Her recent feelings of despondency were intertwined with the failure of her learners to make progress, in sharp contrast to her previous experiences of success.These feelings were exacerbated by negative reactions from the educational authorities and the media, coupled with her frustration at not being heard.

South Africa as an emerging economy market is making progress in the economic sphere, but the quality of education and the unequal distribution of wealth and resultant poverty in certain sectors of the country act as serious inhibitors of further economic growth.The poor quality of education persists despite the fact that change has become integral to the South African education system.Numerous policy changes and subsequent change initiatives on school level have not resulted in sufficient progress.We argue that one of the reasons for the weak effect of education reforms is that the important role of teachers' identities as they are forced to position themselves within new approaches to and practices in education is not sufficiently considered.According to Geijsel and Meijers (2005) identity learning is necessary during periods of innovation and change.They claim that without changes in teachers' personal identity sustainable changes in schools will not be possible.

The OECD report (2013) suggests that the better use of available resources in education would enhance the quality of education and make a positive contribution to economic progress in South Africa.As teachers form the bulk of (human) resources in education and mostly carrying the responsibility for the implementation of change initiatives, it seems reasonable that the nurturing and support of teachers should carry high priority.Iris's message endorses the need of teachers to be heard, to be acknowledged for what they accomplish under difficult circumstances, and highlights their dire need for support.Teachers need access to tools that afford their agency and active stance.The right balance between monitoring and support is crucial, while providing more support staff to schools would allow teachers more time to spend on teaching (OECD, 2013).These messages need to be heard by those in power if a true and enduring transformation in education is to be effected.