Sole reliance on one drug, Praziquantel, for treatment and control of schistosomiasis raises concerns about development of widespread resistance, prompting renewed interest in the discovery of new anthelmintics. To discover new leads we designed an automated label-free, high content-based, high throughput screen (HTS) to assess drug-induced effects on in vitro cultured larvae (schistosomula) using bright-field imaging. Automatic image analysis and Bayesian prediction models define morphological damage, hit/non-hit prediction and larval phenotype characterization. Motility was also assessed from time-lapse images. In screening a 10,041 compound library the HTS correctly detected 99. 8% of the hits scored visually. A proportion of these larval hits were also active in an adult worm ex-vivo screen and are the subject of ongoing studies. The method allows, for the first time, screening of large compound collections against schistosomes and the methods are adaptable to other whole organism and cell-based screening by morphology and motility phenotyping.
Infection with parasitic worms (helminths) causes a huge burden of human disease [1] and economic loss to the livestock industry [2]. Currently the major control strategy for the human diseases is by large scale drug administration to schools or by mass drug administration [3]. However, the drugs available are limited in number and efficacy and their increasing use worldwide raises concerns about the development of drug resistance [4], [5]. Schistosomiasis affects an estimated 600 million people [6] but only one drug, praziquantel (PZQ), is commercially available for its treatment and control. PZQ is poorly effective against the immature worms [7] and its increasingly widespread use [8] fuels concerns about drug resistance developing [9]. There have been sporadic reports of treatment failures with PZQ [10], [11], [12] and strains isolated from such cases show lower susceptibility to PZQ [13]. However, since the development of PZQ [14] there has been limited interest in discovery of new schistosomicides apart from the recent identification of oxadiazole-2-oxides as lead compounds [15] and of anti-schistosome activities for some anti-protozoal drugs [16], [17]. The approved anthelmintics invariably were discovered by in vivo screening in animal models. However the low throughput and high costs of these models limits the discovery of new anti-helminth agents. Therefore new high-throughput, in vitro phenotypic screening methods are necessary to advance the discovery of new anthelmintics including anti-schistosome compounds. In the recent past small, focussed, compound collections have been screened against adult schistosomes recovered from rodents [18], [19]. To facilitate screening of larger compound collections microplate-based visual assays were developed using in vitro-derived larval stages, schistosomula, which can be generated in very large numbers [20], [21]. With a view to standardization and automation methods other than manual visual assessment have recently been applied to evaluate drug-induced damage to schistosomula [21], [22], [23], [24]. However bright-field microscopy is simpler to set up, reveals drug-specific morphological effects, and is 100% effective in detecting compounds active in the adult ex-vivo assays [20], [21]. To overcome the need for visual assessment we have developed a label-free, high content screen (HCS) using automatic bright-field image analysis to establish and validate a high throughput screen (HTS) for primary drug screening against schistosomes. Compound efficacy is assessed by a combination of larval motility and larval morphology quantified by Bayesian analysis. The methods make it feasible for the first time to screen very large compound collections against schistosomes and are applicable to other larval helminths.
Experimentation was carried out under the United Kingdom Animal' s Scientific Procedures Act 1986 with approval from the London School of Hygiene and Tropical Medicine Ethics committee. CD1 mice supplied by Charles River, UK were maintained at St Mary' s Hospital, Imperial College London. Schistosoma mansoni was maintained by routine passage and schistosomula were prepared and cultured in M169 [25] as previously described [21]. Adult worm ex-vivo drug testing was as previously described [19]. The reference anti-schistosome compounds praziquantel (PZQ) and dihydroartemisinin (DHA) were obtained from Sigma-Aldrich (UK), methylclonazepam (MCZ) and Ro15-5458 (Ro15) were a gift from Dr H. Stohler (Hoffman-La Roche, Basle, Switzerland), oxamniquine (OX) was from Pfizer Ltd (Sandwich, UK) and oltipraz (OPZ) from WHO Special Programme for Research and Training in Tropical Diseases (WHO-TDR; Geneva, Switzerland). Compounds were dissolved in DMSO (Sigma-Aldrich, UK). A 10,041 compound library comprising lead-like compounds was provided by the Division of Biological Chemistry and Drug Discovery, University of Dundee. The last two columns of each test plate were reserved for controls. The test compound solvent, DMSO, was used as the negative control and added to 16 wells. Our initial testing of the image analysis models revealed that OPZ induced the lowest phenotype and motility scores reflecting the visual assessment that OLT caused the most severe effects of all of the anti-schistosome compounds tested. Therefore, OLT was chosen as the positive reference standard and applied to 8 wells. PZQ, the current therapy for schistosomiasis, induced intermediate phenotype and motility scores and so 4 wells of PZQ were included on each plate as an arbitrary check on plate performance. Black 384-well clear-bottomed plates were selected for imaging (PerkinElmer, UK Cat no 6007460). Into each well 0. 5 µl of test compound or DMSO was dry stamped using the Biomek FXp (Beckman Coulter, High Wycombe, UK). When necessary a prior intermediate dilution step in DMSO was carried out in V-bottomed dilution plates (Greiner bio-one, UK, cat no 781280). Schistosomula (120/well) were added to each well in 80 µl of M169 media using a Matrix WellMate (Thermo Scientific, Basingstoke, UK). Plates were then incubated in a Cytomat C2 automatic incubator (Thermo Scientific, UK) at 37°C, 5% CO2 for 3 days. A Scara Robot (KiNEDx Robot KX-300-470, Peak Robotics, Colorado, USA) controlled by Overlord 3 (Process Analysis and Automation, Hemel Hempstead, UK) was used for all plate movements. After 3 days culture schistosomula were redistributed and disaggregated by using the Biomek FXp programmed to aspirate and dispense 40 µl of the well contents in each of the 4 corners of each well (×3). Bright-field images were collected using an ImageXpressMicro HCS microscope (IXM; Molecular Devices, Wokingham, UK) fitted with a PhotometricsCoolSnapHQ camera (Roper Scientific, Germany). Focussing of the plate and well bottom was achieved by the IXM high-speed laser auto-focus, with a 25 µm offset to focus on the larvae. For motility analysis 5×6 sec interval time-lapse images were collected using a 4× S Fluor 0. 2NA Nikon objective. For detailed morphology a 10× Ph1 Plan Fluor DL 0. 3NA Nikon objective was used to collect 4 adjacent images, which were tiled together to maximise larval numbers for phenotype analysis. After imaging, the plates were visualized by two independent assessors using an inverted microscope (LeitzDiavertWetzlar, Germany). Differences in phenotype and motility scores were measured by one-way ANOVA with a Dunn' s post-test to measure significant differences between DMSO control wells and individual drug treatments. Z factors for both the phenotype and motility scores were measured on a per plate basis in Pipeline Pilot 8. 5 (Accelrys Inc. , San Diego, USA) with an acceptable score being >0. 5 [26].
Following preliminary assessment of appropriate screening concentration/hit rate, the 10,041 compound library was screened at 10 µM. All of the plates were also visually scored by two independent assessors [21]. Using the HCS hit thresholds defined above, all the visual hits (379) apart from four were determined to be hits by the automatic analysis (Figs. 5A & B). Three of the failures were ascribed scores which fell just outside the hit threshold and one failed to segment due to the parasites remaining aggregated. The hit region also contained 109 wells which were visual non-hits (i. e. false positives by HCS). Of these, 86 were wells containing compound crystals. All of these were readily rejected as hits on manual review of the corresponding images in the automated HCS plate reports by marking the “Non-Hit checkbox” (Fig. S3E). Overall, during the manual plate reading, 780 wells were found to have crystals, of which 130 were deemed to be hits. Importantly, all of these fell in the HCS hit region. A novel phenotypic effect (internal vacuolation) was also identified during visual assessment/plate reporting for a number of compounds, a proportion of which were scored as hits by the HCS. Visually, larval viability was not considered sufficiently reduced to designate these as hits and none of the compounds were active in the adult assay. Z factor scores were reviewed to assess plate performance during screening (Fig. 5C) all of which were within an acceptable range. Hits from the screen were also analysed and grouped according to larval phenotype by the Bayesian categorization model to determine which anti-schistosome compound they most resembled. From the 378 hits, 175 were ascribed to the OPZ treatment class, 60 to PZQ, 13 to MCZ, 83 to Ro15,34 to OX and 13 to DHA. The assay was further validated by re-testing a selection (796) of hits and non-hits from the 10,041 compound library along with compounds from the WHO-TDR set. There was a high level of concordance between the initial and repeat testing (92. 3% for morphology, Fig. 6A; 95. 2% for motility, Fig. 6B). In vitro testing against ex-vivo adult worms is a crucial secondary screen since the adult worm is the key target of drug action. Preliminary testing of a few of the larval hits in the secondary adult worm assay [19] at 10 µM yielded a very low hit rate and so the hits were all tested at 20 µM which gave 45 adult hits. Plotting the larval phenotype and motility scores for these hits (Fig. 7) showed that the majority corresponded to severe larval phenotypes but a few were scattered throughout the hit threshold region. The number of hits ascribed to different treatment classes were OPZ 28, PZQ 6, DHA 4, Ro15 4, OX 2 and MCZ 1. Subsequent IC50 testing of the adult hits identified 7 compounds which had IC50s of <10 µM and which are the subject of on-going studies. These compounds were attributed the drug treatment classes OLT 5, PZQ 1, OX 1.
Whole organism screens have an advantage over more target-based approaches as hit compounds can be directly translated into new therapeutics and it has been suggested that the lack of these screens has impacted on the discovery of new compounds [28]. We have established the first HTS for whole organism screening of helminths based on bright-field HCS analysis of morphological and motility changes in schistosome larvae. The published examples of primary high content drug screens are predominately cell-based [29], [30]. A few involving whole organism screens for Caenorhabditis elegans [31], zebrafish embryos [32], Leishmania major [33] and Plasmodium falciparum [34] have been reported. These exploit use of fluorescent probes or proteins since fluorescence provides more contrast, sharpness and discrimination compared with transmitted-light imaging. However, use of fluorescent probes often involves more manipulations and use of potentially toxic fluorophores [35]. Furthermore, fluorescent transgenic lines are not available for certain organisms of interest including parasitic helminths. Transmitted-light imaging and analysis has been developed for whole well segmentation of C. elegans [36] and to demonstrate drug-induced motility changes using optical flow in adult Brugia malayi [37]. Our approach focussed on the development of bright-field imaging of morphology and motility of larval schistosomes directly comparable to the manual visualization used previously [20], [21]. This requires segmentation and analysis of whole organisms as has been described for C. elegans [31], [38], [39]. Our segmentation method differs from previously reported approaches applied to schistosomula, which used a single threshold and aimed to segment touching larvae [40], [41]. The protocol we describe uses an adaptive threshold (relative to regional background intensity) and avoids the need to segment touching organisms due to successful larval resuspension prior to imaging. This resulted in capture of sufficient individual larvae (≥10/well) at 4× and 10× for analysis in 97. 8% wells from the 10,041 compound library. Analysis of cells or whole organisms by HCS has the potential to generate image profiles to define characteristic drug phenotypes [42] which may ultimately be interpretable in relation to compound activity, modes of action and molecular targets, currently undefined for any of the known schistosomicides [43]. Different schistosomicides induce a range of distinct morphological effects in both adults [19] and larvae [20] which led us to develop the treatment class model, grouping test compounds causing similar effects to the anti-schistosome drugs. In screening the 10,041 compound library, larval treatment classes ascribed to adult hits with IC50<10 µM were OLT 5, PZQ 1, OX 1. Using an alternative approach to our Bayesian treatment class model, agglomerative hierarchical clustering or DBSCAN has been recently developed [41] to ascribe larval phenotypes according to defined morphological classes. The utility of such classifications models according to phenotype may become clear as more hits are detected and structure/activity relationships are understood. The Bayesian models developed here can also be readily modified by uploading larval images of any novel phenotypic effects identified during image review for any compounds of particular interest e. g. those which show adult worm activity. Similarly the models could be modified by addition of images of novel phenotypes deemed on manual review not to warrant hit status e. g. those causing internal vacuolation identified during our screening. The HCS was validated using several compound collections. At 10 µM, a commonly used concentration for primary screening, the models reliably and reproducibly distinguished all of our reference schistosomicides from the controls with the exception of DHA and Ro15. This is not considered a failure of the primary assay since both of these compounds are inactive in the visual larval and adult worm in vitro assays at 10 µM. Based on the results with the anti-schistosome drugs, hit thresholds of −0. 15 for phenotype and −0. 35 for motility were established and proved robust in detecting 40 previously tested adult hit compounds [19] and 99. 8% of visually assessed hits from the 10,041 compound library. The HCS produced a false positive rate of 1. 1%, mostly (78%) due to the precipitation of test compounds in wells which in fact contained healthy parasites and which were readily redefined as non-hits during routine reviewing of the automated plate reports. The HCS offers significant advantages over other recent approaches to objective quantitation of schistosomula damage. Peak et al. (2010) [23] successfully assessed severe drug effects based on uptake of fluorescent markers but the assay involves multiple wash steps and was unable to detect more subtle effects e. g. caused by PZQ. An assay based on use of the redox indicator, Alamar Blue, was similarly less sensitive than visual assessment for more subtle effects and was influenced by variation in parasite numbers per well [21]. Isothermal microcalorimetry [44], assessment of motility via electrical impedance measurement [45] and optical flow [37] are also able to quantitate drug effects in schistosomes, but are not currently readily adaptable to high throughput applications. Much of the HTS described is automated and simple to operate. Once test plates have been set up and left for 3 days in the Cytomat incubator, the image capture and analysis systems would run automatically after opening “Overlord 3”, the automation control software and pressing “run”. Thereafter, plates emerge from the Cytomat and are robotically moved around for barcode reading, parasite resuspension, imaging and then return to the cytomat. Subsequent analysis involves opening Pipeline Pilot 8. 5 and running the analysis protocol. Once complete the plate reports are accessible within an intranet web-port. To become sufficiently familiar with the current customized system would require only a couple of days of training and completing a few test runs. Basic modification of the analysis protocols e. g. to run fewer test compounds in a plate, would require some familiarity with Pipeline Pilot 8. 5 as well as MetaXpress software which controls the IXM microscope. More significant alterations of the protocols would require in-depth knowledge of all the different software involved. Currently the platform takes 2 hrs to image a 384 well plate and a further 2 hrs to analyse the phenotype and motility images. The system can be programmed to start imaging at any time of the day and could run close to continuously. So if test plates were set up on each of 4 days per week, the throughput, limited by plate reading, would be ∼48 plates or 16,896 compounds/week which would require around 2×106 cercariae/week. In fact it is cercarial production which is limiting our current throughput capability to around 10 plates twice per week (∼7,000 compounds/week, 350,000/year). In conclusion, the HCS described is suitable for primary screening of large compound collections for activity against schistosomes. Further studies are ongoing to adapt this system to screen against several species of nematode larvae of medical and veterinary importance, which may allow parallel testing of libraries against various helminths.
Schistosomiasis is a severe helminth infection affecting an estimated 600 million people. The one drug widely available, praziquantel (PZQ), is not ideal. PZQ kills the adult worms but not the developing juveniles so the treated patient may not be cured long-term. In addition, use of repeated mass treatment campaigns with PZQ to control morbidity raises concerns about the development of drug resistance. Our work is aimed at providing starting points for drug discovery programs for schistosomiasis by screening large compound libraries against whole organisms. Praziquantel and several other known anti-schistosomal drugs are also active in vitro against the adult worms and the larval stages, schistosomula. The latter are ideal for novel drug screening as they can be produced in large numbers in vitro, are small and so are amenable to screening in microwell plates. Drug activity can be assessed visually but this is subjective and laborious. We have built an automated system for assessing drug action involving the collection of images of the larvae and the development of computer algorithms to analyze their morphology and motility, defining them as" hits" or" nonhits." The method is reliable, consistent and efficient, making it feasible, for the first time, to screen large compound collections.
biotechnology
medicine
drug research and development
science policy
technology development
drugs and devices
pharmacology
biology
zoology
drug discovery
helminthology
Whole Organism High-Content Screening by Label-Free, Image-Based Bayesian Classification for Parasitic Diseases