Abstract Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

Author Summary New drugs for visceral leishmaniasis, are urgently required as existing drugs have serious shortcomings including toxicity and drug resistance. This disease is caused by parasites from the Leishmania family which live inside human cells. Screening large collections of chemicals (>100,000) to identify compounds that kill parasites has been used to identify new start points for drug discovery. It is complex and expensive to look at such numbers using intracellular parasites. To circumvent this, many groups screen using parasites adapted to grow outside human cells (axenic forms). However, the established protocols identify growth slowing compounds as well as compounds that kill parasites. Cytocidal compounds are better start points for drug discovery. Here we present a screening cascade based on a modified axenic Leishmania assay adapted to identify compounds that kill parasites. We show that these compounds have a higher probability of being active against intracellular parasites. This new screening cascade was used to screen a compound collection and led to the identification of two new chemical series with antileishmanial activity. Their activity was confirmed against intracellular parasites. They are potential candidates for further drug development. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery.

Citation: Nühs A, De Rycker M, Manthri S, Comer E, Scherer CA, Schreiber SL, et al. (2015) Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity. PLoS Negl Trop Dis 9(9): e0004094. https://doi.org/10.1371/journal.pntd.0004094 Editor: Gerald F. Späth, Pasteur Institute, FRANCE Received: March 26, 2015; Accepted: August 30, 2015; Published: September 25, 2015 Copyright: © 2015 Nühs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was primarily funded by the Drugs for Neglected Diseases initiative (www.dndi.org). JRI is an employee of DNDi and helped with the study. In particular, he secured access to exemplars of compound from DNDi's drug development portfolio for benchmarking the new assay. For the work described in this paper, the Drugs for Neglected Diseases initiative received financial support from the following donors: Department for International Development (UK), Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF; Germany), Bill & Melinda Gates Foundation (US), Médecins Sans Frontières (Doctors without Borders), Dutch Ministry of Foreign Affairs (DGIS; The Netherlands) and Swiss Agency for Development and Cooperation (SDC; Switzerland). The donors had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This study was funded in part by a grant from the Bill & Melinda Gates Foundation (Grant OPP1032518 to SLS) and by a Wellcome Trust Strategic grant (100476/Z/12/Z to DWG). The Bill & Melinda Gates Foundation and Wellcome Trust 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.

Introduction The protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a group of diseases that is prevalent in 98 countries and 3 territories with approximately 1.3 million new cases occurring annually. There are estimated to be 20,000 to 40,000 deaths per year [1]. Leishmaniasis occurs in three main forms. Visceral leishmaniasis is the most severe form, where the parasites migrate to the internal organs, particularly the spleen and liver, resulting in death if untreated [2]. Cutaneous and mucocutaneous leishmaniasis are dermal infections which carry lower mortality but are highly disfiguring. The associated social stigmatization can have significant negative effects on psychological well-being [3, 4]. Currently, treatment of visceral leishmaniasis is limited to a few drugs used either in monotherapy or in combination. For reasons still to be understood, some of the treatments show a lack of clinical efficacy in certain geographic regions, notably in East Africa. In India and Nepal the emergence of resistance to antimonial therapy also limits the treatment options. Additionally, the occurrence of moderate to severe adverse effects, the existence of contraindications such as pregnancy in the case of miltefosine, the relatively high cost of the treatments as well as the logistical complexity related to the storage and use of drugs in the endemic regions further restrict the use of amphotericin B, miltefosine, antimonials and paromomycin. There is therefore an urgent need for new and better drugs to address treatment needs [5–7]. High-throughput screening of diverse compound sets in phenotypic assays has proved an effective way of discovering new start points for drug discovery [8, 9]. To facilitate screening of large compound collections against Leishmania, axenic amastigotes have been used as a surrogate for the disease-causing intracellular form [10–12]. Axenic amastigotes are thought to be a more relevant model of the human lifecycle stages of leishmania infection in comparison to the promastigote [13–17]. Several assays using axenic stages of Leishmania have been developed to the scale and robustness appropriate for library screening. However, we and others have questioned the relevance of these assays due to the poor translation of many axenic hit molecules into the physiologically more relevant but more complex intracellular Leishmania assays [10, 18–22]. The poor confirmation rate of promastigote and axenic amastigote active compounds in intracellular assays could result from the many differences between the assays, including the localisation of intracellular parasites in the more difficult to access parasitophorous vacuole, differences in pH and composition of the growth media, stage-specific differences such as alternate energy pathway usage [16, 23, 24], etc. Insights derived from our work with Trypanosoma brucei [25] point to another potential factor: many published axenic assays are likely to report not only cytocidal compounds but also growth slowing and cytostatic compounds. The latter two types of compounds are unlikely to show activity in intracellular assays which, due to the slow replication rate of the intracellular parasites and sensitivity of high content readers, tend to only report cytocidal molecules. In this paper we report the development and validation of a novel axenic amastigote Leishmania assay that reports only cytocidal molecules. This new assay was validated using a diverse compound library previously screened in our historic axenic format and in the intracellular assay [18]. We also used the new axenic assay as a primary screening platform to screen a diversity-oriented synthesis library [26]. We identified two new chemical series with antileishmanial activity and show that the new axenic assay has a significantly improved translation to the intracellular assay. We propose that the novel axenic assay provides Leishmania drug discovery efforts with an improved high-throughput platform for the screening of large compound libraries as it does not suffer from the high false positive rates seen in other axenic assays and provides both higher throughput and better robustness than intracellular assays.

Methods Parasite strain and maintenance Leishmania donovani BOB cells (LdBOB) used in this study are a cloned line from strain MHOM/SD/62/1S-CL2 [13]. Cultures were maintained as described previously [18]. Materials White, clear-bottom assay plates (384-wells) were obtained from Greiner (historic axenic assay) and Corning (novel axenic assay). Echo plates were obtained from LabCyte. Chemicals Amphotericin B, Dimethyl Sulfoxide (anhydrous, ≥ 99.9%, DMSO) and Resazurin were obtained from Sigma. The small diverse library used in this study contained 15,667 compounds, dissolved at 10 mM in DMSO and stored under low-oxygen and low-humidity conditions. The design of this diverse library is described elsewhere [27]. The Diversity-Orientated Synthesis (DOS) Informer Set (9,907 compounds) is a subset of the Broad Institute’s diversity-oriented synthesis library, which comprises approximately 100,000 structurally diverse small molecules that combine the complexity of natural products and the efficiency of high-throughput synthesis [26, 28, 29]. These small molecules have a higher ratio of sp3-hybridized atoms and stereocentres relative to compounds found in conventional screening collections [30]. The compounds are structurally diverse, from >30 different individual libraries and >250 unique scaffolds. Moreover, where possible, all stereoisomers have been individually synthesized, providing rich stereo-structure-activity relationship (SSAR) data directly from primary screens and facilitating rapid prioritization and optimization of hit compounds. Compound handling Compounds were dispensed into 384-well assay plates by acoustic dispensing (LabCyte ECHO). For potency determinations, ten-point one in three dilution curves were generated, with a top concentration of 50μM. Potencies are reported as pEC 50 (-LOG(EC 50 [M])). Data analysis All data was processed using IDBS ActivityBase. Raw data was converted into percent inhibition through linear regression by setting the high inhibition control as 100% and the no inhibition control as 0%. For primary single concentration screening in the novel axenic assay we introduced a static control as 0% inhibition (signal at time of assay start), which is explained in more detail in the results section. Potency plates were normalised to DMSO control (0% effect) and 2μM amphotericin B control (100% effect). Quality control criteria for passing plates were as follows: robust z’ ≥ 0.5, signal to background > 3,% coefficient of variation for 0% inhibition controls < 15. The formula used to calculate robust z’ is 1-((3 x (1.4826 x MAD [0% inhibition controls] + 1.4826 x MAD [100% inhibition controls]))/(Median [0% inhibition controls]—Median [100% inhibition controls]), with MAD the median absolute deviation. The formula used for signal to background is: Median [0% inhibition controls] / Median [100% inhibition controls]. Curve fitting was carried out using the following 4 parameter logistic equation: y = A + (B - A) / (1 + ((10C) / x)D), where A =% inhibition at bottom, B =% inhibition at top, C = 50% effect concentration (EC 50 ), D = slope, x = inhibitor concentration and y =% inhibition. For compounds with low activity and poor definition of the curve top, B was fixed to 100. For the determination of the reference compound panel potency, all experiments were carried out with a minimum of three independent repeats. Limit of detection (LoD) Serial dilutions of LdBOB cells with defined cell concentrations were made. Each of the resulting concentrations was dispensed into at least 24 wells of a 384-well plate (50 μl and 25 μl per well for determining the LoD of the historic and novel axenic assay respectively), the rest of the wells contained media only (blank). The read-out for the historic axenic assay was performed by adding resazurin at 0.05 mM final concentration followed by incubation for 4 h at 37°C and 5% CO 2 . Fluorescence intensity was then measured using a Perkin Elmer Victor 3 plate-reader (excitation 528 nm, emission 590 nm). The read-out for the novel axenic assay was carried out by adding BacTiter-Glo (Promega) (volume equal to culture media volume) to each well and the luminescence was immediately read in a Victor 3 plate-reader. A linear regression was fitted and the LoD was derived as the number of cells equal to the mean signal of the blank wells plus 3 times the standard deviation. The values were determined from 4 independent experiments LoDs are reported as LoD +/- Standard Deviation (StDev). High-throughput assays Historic axenic assay. The assay conditions are described previously [18]. Briefly LdBOB amastigote-like cells were seeded at 250 cells per well in a 384-well plate containing the compounds. After a 68 h incubation at 37°C and 5% CO 2 , resazurin was added at 0.05 mM final concentration. Plates were incubated for a further 4 h and then fluorescence was measured (excitation 528 nm, emission 590 nm) with a plate reader. Intramacrophage assay. The assay conditions are described previously [18]. Briefly PMA differentiated THP-1 cells were infected overnight with eGFP expressing LdBOB amastigote-like cells at a multiplicity of infection of 5. Next, any remaining free amastigote-like cells are removed and compounds are added. The microscopy-based read-out is done after a four day compound incubation. HepG2 assay. The assay conditions are described previously [31]. Briefly HepG2 cells were incubated for 72 h with compounds, followed by a resazurin-based read-out (fluorescence, excitation 528 nm and emission 590 nm) with a plate reader. Novel axenic assay. Test compounds were pre-dispensed into white 384 well plates (Corning). For library screens one compound per well was tested at the indicated final concentration. The following controls were included on the plates: maximum effect control: Amphotericin B (final concentration 2 μM), ATP contamination control: media (± DMSO), cell growth control: DMSO, zero percent effect control: cells at starting concentration. The zero percent effect control wells are left empty during the course of the assay. Media only was dispensed into the ATP-control wells followed by dispensing LdBOB axenic amastigote-like cells at 2 x 104 cells / well to the rest of the wells by using a Wellmate Microplate Dispenser. The assay volume is 25 μl / well. After 72 ± 3 h at 37°C and 5% CO 2 LdBOB axenic amastigote-like cells are added to the zero percent effect control wells at 2 x 104 cells / well followed by read-out with “BacTiter-Glo Microbial Cell Viability Assay” from Promega according to manufacturer’s instructions. Plates were then sealed with clear film and relative luminescence was detected using a plate reader (Victor 3) from Perkin Elmer or PHERAstar FS from BMG LABTECH, with 0.5 s detection time per well). Potency determinations were carried out as library screens with the exception of the following: ten-point curves with one in three dilutions were generated with a top concentration of 50 μM.

Discussion The need to identify new candidates entering drug development for visceral leishmaniasis is high considering the low number of compounds currently progressing through the R&D pipeline for this disease [32]. In many disease areas phenotypic screening of large diversity-oriented compound collections has been successful at identifying novel active starting points [8, 9, 33, 34]. For leishmaniasis, finding new start points is hampered by the lack of predictive, high-throughput compatible assays. When compounds are identified, they suffer from the intrinsically high attrition rate associated with early-stage discovery and lead-optimisation programmes [35]. It is therefore critically important to find new approaches to fill the leishmaniasis pipeline. The intramacrophage amastigote Leishmania model is currently considered the gold standard for in vitro drug susceptibility characterization [17, 18, 22, 36, 37]. Running this assay at a sufficiently high throughput to screen medium- or large-size compound collections is technically as well as financially challenging due to the complexity and limited robustness of this type of assay. While axenic parasites (promastigotes or amastigotes) can be used readily in high-throughput assays, the relatively poor translation into the intracellular assay limits their usefulness [17, 18, 21, 38]. In this work we set out to develop a new Leishmania in vitro screening cascade that is suitable for high-throughput screening so that large compound collections can be accessed. As a primary screening platform we developed a new high-throughput axenic amastigote assay with significantly improved predictivity of intramacrophage activity. We have achieved this by altering the assay conditions so that only cytocidal compounds are identified. Our results show that the main reason for the poor correlation between the old axenic assay and our intracellular assay is assay setup related, rather than the result of differences in biology between the different stages (~ 80% of hits identified in our historic axenic assay were not cytocidal). While we cannot be sure this is the case for other published axenic assays as detection limits are rarely reported, we expect this may apply broadly, as a side-effect of using fast growing organisms in combination with relatively low sensitivity read-outs such as resazurin. In view of the low hit rate for Leishmania that we and others have found, there is a concern around potential false negatives. While these clearly exist, many are actually toxic to the host cell in the intracellular assay and can be considered intracellular assay false positives. We have shown that the root cause for approximately half of the non-toxic false-negative compounds that we tested is the difference in concentration that we used to screen in the axenic compared to the intracellular macrophage assay (Fig 2). As most of the remaining false-negatives are weak in the intracellular assay it is possible that they may show activity in the novel axenic assay when tested at higher concentrations. Interestingly, there are a number of compounds that are active in the novel axenic assay and not active in the intracellular assay. These may represent compounds that hit targets that are not essential intracellularly, perhaps as a result of differences in energy metabolism or supply of essential metabolites. However, our preliminary analysis suggests that these may cluster in a physicochemical space that may preclude their passage through cell membranes under physiological pH. Such compounds may represent novel start points for drug discovery, if their permeability can be improved while maintaining activity. Testing a panel of previously identified antileishmanials showed good correlation between the novel axenic assay and the intramacrophage assay for most compound classes. The aminoquinolines (mefloquine & amodiaquine) were only active in the intracellular assay, presumably due to their charge at acidic pH, resulting in lack of permeability in the novel axenic assay (pH 5.5) and lysosomotropic accumulation in the intramacrophage assay [39]. Surprisingly, miltefosine was around ten-fold more active in the intracellular assay relative to the novel axenic assay. We and others have previously observed similar activity in axenic and intramacrophage models for miltefosine [17, 18]. Miltefosine is actively taken up by Leishmania [40, 41], and therefore it is plausible that the potency difference is due to the much higher cell density in the novel axenic assay compared to our historic assay (80-fold higher), as this could result in lower miltefosine concentrations in the cells and hence lower potency. Disulfiram was only active in the novel axenic assay. Due to technical differences between the two assays there is a higher chance of compounds precipitating in the intracellular assay, and this could have been a problem with disulfiram as its aqueous solubility is poor and in addition this compound is unstable in serum [42]. The activity seen for VL-2098 in the novel axenic assay is in line with recently published data [43], however the activity against intracellular amastigotes is lower than expected from the publication. A second sample of this compound was tested and gave the same results. A potential explanation for this discrepancy is the poor solubility of this compound [44]. Overall the results from this panel of compounds shows that there is a good correlation between our novel axenic assay and our intramacrophage assay, and that almost all compounds with proven antileishmanial activity show activity in the novel axenic assay, supporting its use as a primary screening assay. We propose that the novel axenic assay is the most suitable primary assay for a screening cascade aimed at accessing large compound collections as it combines the throughput and robustness of axenic assays with good predictivity of intramacrophage activity. Hits from the single-point screen should be tested in potency mode both in the axenic assay and in a human cell counterscreen to rule out any toxicity (ideally targeting a 10-fold or higher selectivity window between the two assays). Finally, the active and selective compounds should be tested in the intracellular model (cascade shown on Fig 4). We validated the use of this cascade by screening a set of ~10,000 DOS compounds and identified two active chemical series of interest. This screen confirmed further that the novel axenic assay is a suitable primary screening platform as 92% of the confirmed actives also showed intracellular activity. The two most interesting hit series incorporate a densely functionalized azetidine ring system each containing three stereogenic elements. While azetidine-related systems such as β-lactams have played an important role in drug discovery, the fully reduced form such as those in our two series of interest have been significantly less studied. These libraries were prepared as part of a collection of skeletally diverse azetidine-based scaffolds. An important feature of this compound collection is that it contains stereochemical diversity and in our experience, Stereochemical Structure Activity (SSAR) analysis of a hit compound can give an indication on how selective and specific its interaction is with its molecular target [45]. While two stereoisomers of BRD6650 were active, the majority of the stereoisomers tested had at least a 9-fold drop in potency indicating a selective and specific interaction with a molecular target. The two series of interest differ in terms of their substituents at R1 and R2 and also the substitution pattern of the phenyl group at C3 which is para in series 1 but ortho in series 2. Synthesis of additional analogues to further investigate SAR and profiling of the current leads in in vitro ADME/PK would allow us to assess the potential for these series for therapeutic development. In summary, we have developed and validated a novel axenic Leishmania assay that specifically identifies compounds with a cytocidal mode of action. This new assay has been profiled in terms of its ability to predict activity in the intracellular amastigote Leishmania stage which is currently considered the gold standard for in vitro drug screening for visceral leishmaniasis. We have demonstrated that this new assay is suitable for primary screening and provides a useful method of triaging compounds to significantly reduce the number of compounds to be profiled and confirmed for activity through the more complex intracellular assays. Additional new active hit series against Leishmania donovani are expected to be identified from several ongoing high-throughput screening campaigns incorporating the novel axenic assay as a primary screening tool. Overall, our findings show that assay conditions play a significant role in the nature of the compounds identified (i.e. growth slowing vs cidal) and demonstrate that appropriate axenic assays, particularly in the context of large-scale drug-discovery, can be relevant and of great value.

Supporting Information S1 Text. Includes the following items: In tabular form-(Table A) Hit-rate and Control definitions in Historic, Novel and Intramacrophage Assay and (Table B) potencies for reference compound panel.In figure form—effect of assay start day (Fig A), different cell stocks (Fig B) and cell passage on assay performance (Fig C); reproducibility of compound potency across assay replicates (Fig D) and a representative plot from the Broad's SSAR viewer tool (Fig E). https://doi.org/10.1371/journal.pntd.0004094.s001 (DOCX)

Acknowledgments We would like to thank DDU Compound Management for preparation of the screening plates, and the DDU database managers for help with registration and data processing.

Author Contributions Conceived and designed the experiments: AN MDR JRI DWG. Performed the experiments: AN SM. Analyzed the data: AN MDR EC CAS SLS JRI DWG. Contributed reagents/materials/analysis tools: SLS. Wrote the paper: AN MDR EC CAS SLS JRI DWG.