Drug discovery for schistosomiasis: hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening - PubMed (original) (raw)
Drug discovery for schistosomiasis: hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening
Maha-Hamadien Abdulla et al. PLoS Negl Trop Dis. 2009.
Abstract
Background: Praziquantel (PZQ) is the only widely available drug to treat schistosomiasis. Given the potential for drug resistance, it is prudent to search for novel therapeutics. Identification of anti-schistosomal chemicals has traditionally relied on phenotypic (whole organism) screening with adult worms in vitro and/or animal models of disease-tools that limit automation and throughput with modern microtiter plate-formatted compound libraries.
Methods: A partially automated, three-component phenotypic screen workflow is presented that utilizes at its apex the schistosomular stage of the parasite adapted to a 96-well plate format with a throughput of 640 compounds per month. Hits that arise are subsequently screened in vitro against adult parasites and finally for efficacy in a murine model of disease. Two GO/NO GO criteria filters in the workflow prioritize hit compounds for tests in the animal disease model in accordance with a target drug profile that demands short-course oral therapy. The screen workflow was inaugurated with 2,160 chemically diverse natural and synthetic compounds, of which 821 are drugs already approved for human use. This affords a unique starting point to 'reposition' (re-profile) drugs as anti-schistosomals with potential savings in development timelines and costs.
Findings: Multiple and dynamic phenotypes could be categorized for schistosomula and adults in vitro, and a diverse set of 'hit' drugs and chemistries were identified, including anti-schistosomals, anthelmintics, antibiotics, and neuromodulators. Of those hits prioritized for tests in the animal disease model, a number of leads were identified, one of which compares reasonably well with PZQ in significantly decreasing worm and egg burdens, and disease-associated pathology. Data arising from the three components of the screen are posted online as a community resource.
Conclusions: To accelerate the identification of novel anti-schistosomals, we have developed a partially automated screen workflow that interfaces schistosomula with microtiter plate-formatted compound libraries. The workflow has identified various compounds and drugs as hits in vitro and leads, with the prescribed oral efficacy, in vivo. Efforts to improve throughput, automation, and rigor of the screening workflow are ongoing.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Workflow for phenotypic screening of S. mansoni.
The workflow was prosecuted with the Microsource Discovery Inc.'s “Spectrum” and “Killer” collections that together comprise 2,160 (1,992 unique) compounds, including 821 drugs approved for use with humans (
). The goal was to interface this parasite with the 96-well plate-formatted small molecule libraries available at the UCSF Small Molecule Discovery Center (SMDC;
) and common elsewhere, thereby accelerating throughput and facilitating screen automation. Schistosomula are placed at the apex of a three-component workflow that subsequently incorporates screens against adult parasites in vitro and finally an animal model of infection to measure in vivo efficacy. Times at which phenotypes were recorded in vitro are indicated in hours and days Two GO/NO GO workflow filters allow for prioritization of the ‘hit’ compounds in vitro. The numbers of ‘hits’ generated at various points in the workflow are indicated in bold typeface. Data arising from each of the three screening components are posted online as a flat file at The Sandler Center's ‘Low Hanging Fruit” website (
http://pathology.ucsf.edu/mckerrow//fruit.html
), and in a cross-searchable format, at the database maintained by Collaborative Drug Discovery (CDD Inc.; (
http://www.collaborativedrug.com/
). For smaller numbers of compounds, the workflow need not be hierarchically prosecuted, rather every compound can be screened against both schistosomula and adults.
Figure 2. Examples of the different and multiple phenotypes manifest by schistosomula exposed to chemical insult.
Worms were exposed for 7 days to 1 µM of either the schistosomicidal cysteine protease inhibitor, K777 (B), or the current chemotherapy, PZQ (C), and compared to controls with DMSO alone (A). Phenotypes ascribed are ‘dark/dead’ for K777 and ‘overactive/degenerated but mobile’ for PZQ. Images were captured using a Zeiss Axiovert 40 C inverted microscope (10× objective) and a Zeiss AxioCam MRc digital camera controlled by AxioVision 40 version 4.5.0.0 software. Scale bar = 0.2 mm.
Figure 3. Examples of the different and multiple phenotypes manifest by adult S. mansoni exposed to chemical insult.
Worms were exposed for 2 days to 1 µM of either K777 (B) or PZQ (C) and compared to controls with DMSO alone (A). Phenotypes ascribed are ‘slow/sex separated/on sides’ for K777 and ‘slow/dark/shrunken/on sides/sex separated/tegumental blebbing’ for PZQ. Arrows point to female worms. Images captured using a Zeiss 2000-C Stemi inverted microscope mounted over a Diagnostic Instruments Transmitted Light Base and a Zeiss AxioCam MRc digital camera controlled by AxioVision 40 version 4.5.0.0 software. Scale bar = 0.7 mm.
Figure 4. SAR for the phenothiazine and dibenzazepine classes of psychoactive drugs eliciting the ‘overactive’ phenotype in schistosomula.
See Table S4 for individual compounds and Supplementary Videos 1 and 2 to compare phenotypes between ‘overactive’ and control worms.
Figure 5. Structures of compounds prioritized for efficacy tests in the murine model of schistosomiasis mansoni.
(A) Hit compounds arising from the in vitro components of the screen workflow and (B) analogs of niclosamide currently marketed as veterinary anthelmintics or as an intestinal anti-protozoal in humans (nitazoxanide) and tested in the murine model of schistosomiasis.
Figure 6. Effect of lasalocid sodium and anisomycin on male and female worm burdens, and organ pathology (as measured by weight) in mice infected with S. mansoni.
Compounds were administered orally QD or BID at the doses indicated for 4 days 42 days after infection with 140 S. mansoni cercariae. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where _p_≤0.05. Egg burdens were calculated as single values per treatment group (see text for details).
Figure 7. Effect of closantel, oxyclozanide, rafoxanide and PZQ on male and female worm burdens, egg burdens and organ pathology in mice infected with S. mansoni.
Compounds were administered orally for 4 days 42 days after infection with 140 S. mansoni cercariae. Doses administered were: 100 mg/kg BID for closantel and oxyclozanide, 50 mg/kg QD for rafoxanide and 100 mg/kg QD for PZQ. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where _p_≤0.05.
Figure 8. Effect of nitazoxanide on male and female worm burdens, and organ pathology in mice infected with S. mansoni.
Compound was administered orally at the doses indicated for 4 days 42 days after infection with 140 S. mansoni cercariae. Points represent data from individual treated or untreated (control) infected mice. The horizontal bars represent median values. Significance (p) values are indicated where _p_≤0.05. Egg burdens were calculated as single values per treatment group (see text for details).
References
- International Drug Price Indicator Guide. Management Science for Health, Inc; 2006.
- Cioli D, Pica-Mattoccia L. Praziquantel. Parasitol Res. 2003;90(Supp 1):S3–9. - PubMed
- Botros S, Sayed H, Amer N, El-Ghannam M, Bennett JL, et al. Current status of sensitivity to praziquantel in a focus of potential drug resistance in Egypt. Int J Parasitol. 2005;35:787–791. - PubMed
- Hotez PJ, Molyneux DH, Fenwick A, Kumaresan J, Ehrlich Sachs S, et al. Control of neglected tropical diseases. N Engl J Med. 2007;357:1018–1027. - PubMed
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