New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.
The search for new antiparasitic drugs for use in humans has accelerated in the past decade, based partly on the growing recognition that addressing these widespread infections is necessary for poverty reduction. There is a consensus that the drugs available for these pathogens are far from optimal, plagued by susceptibility to resistance, lack of activity against key species (or stages of the life cycle), lack of adequate efficacy in field-compatible delivery regimens, and reliant on single agents for control programmes [1], [2]. Expansion of programs for discovery and development of new compounds has been fueled by investment from donor organizations (such as the Bill and Melinda Gates Foundation and the Wellcome Trust) and increasing participation of the pharmaceutical industry. Some companies have established drug discovery centers for a select set of diseases. For example, the Novartis Institute in Singapore is focusing on malaria, dengue and tuberculosis and the GlaxoSmithKline (GSK) facility in Tres Cantos, Spain, is expanding its efforts to include a number of neglected diseases. We have also witnessed drug donation programmes essential for filariasis control by Merck and GSK as well as praziquantel donation by Merck Serono. This effort has been extended to sharing of proprietary and non-proprietary screening data, exemplified by the recent publication of screening results of corporate compound libraries against malaria parasites [3], [4]. In another development, Novo Nordisk transferred its entire compound library to the National Centre for Drug Screening in Shanghai to support drug discovery for neglected tropical diseases (NTDs) and related capacity building, in collaboration with The Special Programme for Research and Training in Tropical Diseases, at the World Health Organization (WHO/TDR) [5]. WHO-TDR has a long history of drug discovery and development for NTDs [1], [6], [7]. For several decades (1970–2000), it was the primary source of support for systematic antiparasitic screening programmes outside of military institutions and animal health companies [8], [9]. Since then, the creation and evolution of Product Development Partnership (PDP) organizations such as the Medicines for Malaria Venture (MMV), Drugs for Neglected Diseases initiative (DNDi) and Institute for One World Health (iOWH) to support development of promising drugs has become an important factor in bringing modern approaches to pharmaceutical research on neglected diseases [1], [10]. The development of sophisticated antiparasitic drug discovery activities in countries such as India, Brazil, South Africa and China, and increasingly in less developed countries in which these diseases are endemic, introduces new and influential contributors to the renaissance in this area [11]. Furthermore, the continuing efforts of the animal health industry in antiparasitic discovery, particularly in the area of anthelmintics, are being incorporated into human discovery programs [12]; almost all available human anthelmintics were initially developed for use in veterinary settings. Despite these efforts, major gaps in the discovery of new chemical entities for neglected diseases remain, and apart from some repurposed drugs and few new molecules for malaria [13], [14], [15], the international community has not been able to transition novel chemical entities from discovery into development in the past 15 years. An innovation gap has therefore been defined for the various phases of the drug discovery process [16]. Against this background, it is critical to invest in the discovery of new drugs that will contribute in the medium to long term control of these diseases. A network model for drug discovery against multiple neglected diseases has been described as a suitable mechanism to overcome these challenges [1], [16]. This approach involves a coordinated North-South network of collaborators (from both public and private sectors) which operate low- to medium-throughput primary screens against target organisms, supported by medicinal chemistry, drug metabolism and pharmacokinetics resources to kick-start hit-to-lead and lead optimization programs. Over the years part of this screening network funded by TDR has systematically optimized throughput to enable the testing of hundreds to thousands of pre-selected chemicals in diverse collections against the following: Plasmodium falciparum, Leishmania infantum, Trypanosoma brucei, Trypanosoma cruzi, Schistosoma mansoni, Onchocerca lienalis or O ochengi (counterparts in cattle of the human pathogen, Onchocerca volvulus) and Brugia malayi. Higher-throughput methods have been developed for whole organism antimalarial and anti-Chagas screens, both in industrial and non-industrial settings [3], [4], [17]–[20]. However, the throughput capacities of whole-organism screens employing the other pathogens are generally low to medium, permitting parallel testing of tens or hundreds to a few thousand compounds per batch [21]. TDR has taken advantage of the availability of multiple assays available within its network to implement coordinated screening of highly triaged small-to-medium sized libraries against multiple organisms, combined with a rapid assessment of mammalian cell toxicity. Although no in vitro test is fully predictive of in vivo activity, the TDR strategy is generally accepted as standard practice in neglected diseases screens [22], [23]. Consistent with a virtual operation, library evaluation, management and sampling are coordinated with the support of external partners and consultants, who have extensive experience in medicinal chemistry related to drug discovery. These consultants help with prioritization of compound collections to be screened, analysis of the resultant screening hits thorough chemo-informatics analysis and review of available literature on the compound or series identified. Data evaluation, communication and decisions by the drug discovery network are supported by a readily accessible, centralized database in which biological and chemical results from distant sites are uploaded in close to real-time processes for analysis and searching [24]. The database provides chemoinformatics strategies enabling substructure searches to analyze and expand structure-activity relationships (SAR). Here we present the outcome of screening a set of 10,000 compounds against seven (7) NTD pathogens in whole-organism assays through a collaborative network model. The implications of this approach, which we refer to as an integrated multi-pathogen screening strategy, for enhancing drug discovery productivity and efficiency for neglected diseases are discussed.
A library of 10,000 compounds was purchased from Life Chemicals (Ukraine) after extensive review and analysis by medicinal chemists of over 50,000 available diverse structures. The key criteria used in the selection of molecules to be screened are diversity and novelty. To ensure a degree of diversity, selection was enhanced by using the computational informatics tool [Sybyl®, Tripos] [25]. Novelty of compounds for the target diseases was assessed by eliminating previously pursued compounds from in-house screens, available literature and comparison with chemistry already explored for neglected diseases. Based on this triage, a set of over 12,000 molecules was selected for further prioritization by analyzing for the presence of toxicophores and drug-likeness based on meeting at least 4 criteria in the Lipinski Rule of fives [26]. The confirmation by Life Chemical of the availability of close analogs for purchase to develop SAR, the simplicity of synthetic route and the availability in amounts >50 mg in the inventory, obviating the need for re-synthesis to permit testing in animals, was considered as well. The majority of the 10,000 compounds had a high degree of drug-likeness, in accordance with the Lipinski Rule of 5 (Figure 1). Compounds not fully compliant with the Lipinski Rules were included as a significant number of anti-infective and anti-cancer drugs do not meet the full criteria [27], [28]. Molecules were filtered to remove duplicate and reactive groups using Accelrys computational tools [29], to reach the 10,000 unique, non-reactive compound set. Life Chemicals provided the compounds with a special plate partitioning system to cover particular screening and logistics needs for multiple screening of this highly diverse compound set. The compound screening logistics and framework, including sampling, distribution to various screening centers and screening cascade for the various parasites are shown on Figure 2. Standard Operating Procedures (SOPs) were implemented according to established criteria for advancement of compounds from primary screens to more advanced testing in vitro and for efficacy in animal models [16]. The integrated panel of antiprotozoal and anthelmintic screens used in the present study and the standard screening methodologies were adopted as previously described [9], [16], [30]–[35] (see Table 1). Appropriate reference drugs, obtained either from Sigma or WHO-TDR collection, were used as positive controls [9], [16], [30]–[35]: chloroquine sulphate for P. falciparum, amphotericin B for L. infantum, nifurtimox for T cruzi, suramin and pentamidine for T. brucei, praziquantel for S. mansoni, immiticide, amocarzine and ivermectin for O. lienalis or O. ochengi, and ivermectin and diethylcarbamazine for B. malayi. The compound set was tested against the following parasites: intra-erythrocytic forms in human red blood cells of P. falciparum K1 strain (malaria) [9], [16], [32], [33], [35], bloodstream forms of T. brucei Squib 427 strain (human African trypanosomiasis, HAT) [16], [31], [32], [34], intracellular amastigotes of T. cruzi Tulahuen CL2, beta- galactosidase strain (Chagas' Disease) [16], [32]–[35], intracellular amastigotes of L. infantum MHOM/MA (BE) /67 (leishmaniasis) [16], [32]–[36], larval and/or adult worms of S. mansoni Puerto Rican strain (schistosomiasis) [16], [31], [37], microfilariae of B. malayi (lymphatic filariasis) [16], [30] and microfilariae of O. ochengi or O. lienalis (onchocerciasis) [16], [30]. It should be noted that the human pathogen for onchocerciasis is Onchocerca volvulus, but due to lack of a suitable in vitro assay or animal models for this pathogen, O. ochengi and lienalis, which are both animal pathogens, are used in the primary and secondary assays [16], [30]. In some cases, particularly for the antifilarial screens, a second whole organism test with adult stage parasites is included prior to animal evaluation. The decision to utilize microfilariae for primary screening was based on throughput and biological considerations. It is difficult to acquire enough adult filariae to enable screening more than 1000 compounds per year [21], a rate far lower than needed for timely evaluation of the available compounds. In light of this situation, and considering that we are targeting molecules that are active on both stages, microfilariae screening was adopted as a suitable filter to identify compounds with potential macrofilaricidal activity [30]. An important part of the screen is the evaluation of all compounds for cytotoxicity using the diploid human embryonic lung fibroblast MRC-5 cell line (Figure 2). Further evaluation of cytotoxicity also takes advantage of the assays for intracellular protozoan parasites, as in the case of L. infantum and T. cruzi, which include murine peritoneal macrophage host cells [36] and MRC-5SV2 human lung fibroblast cells, respectively, in the culture system. Hits identified from the screens were initially compared with cytotoxicity data for selectivity. Further filtering of the data was done using the published ‘Hit’ criteria for all pathogens combined with classical medicinal chemistry criteria [16]. All screening actives were evaluated for prior art through searches of Chemical Abstracts, and any relationship to compounds in development in related or other therapeutic areas investigated by substructure searches in drugs databases, principally the Investigational Drugs database (IDdb3; now Thomson Reuters Partnering). Substructure searches were also done in the TDR database covering previously tested libraries and medicinal chemistry projects.
From the screen of 10,000 compounds, 744 were identified as active against at least one pathogen (Figure 3). Cytotoxicity data were used to aid selection and prioritization of compounds for progression to the next stage of the lead discovery process. To the best of our knowledge, this is the first time in which comparative data from essentially simultaneous screens against multiple neglected diseases pathogens and cytotoxicity data have been used in an integrated analysis for lead identification. Specific hit series from each pathogen are described below. To further illustrate the results from this multi-pathogen and cytotoxicity screening, a series of case examples with chemical structures covering one or more diseases are presented (Table S1). Identified hits are either referred to as series, consisting of a group of active molecules having similar core chemical structures/scaffolds, or as singletons if only one member of a chemical family is identified as active against a pathogen.
The integrated, multi-pathogen, collaborative approach to the discovery of new chemical entities described here promises to increase efficiency while reducing costs and second-stage attrition in the discovery of high-quality leads compared to operations which target pathogens individually. Although this work focuses on the initial whole organism pathogen screening programme for hit identification, it should be stressed that it is part of an integrated North-South drug discovery platform that includes medicinal chemistry, pharmacokinetics and early toxicology [1], [16]. The approach needs to be scaled up and implemented more broadly, especially in developing countries, to ensure that the diverse set of NTDs receives the attention they warrant. This will require enhanced financial support and commitment from stakeholders as the current funding level is grossly insufficient relative to the burden of these infections on the developing world. In this context, a concerted global effort that interfaces with and strengthens the R&D capacity of institutions in developing countries, for example through ANDI, would greatly maximize resources and ensure that resultant drug leads are efficiently advanced. In support of this approach, some of the hits described in this paper are being transferred to ANDI to support innovation and capacity building in Africa. In addition, the chemical structures for most of the hits from the different pathogens described in this paper can be accessed through the TDR targets database (www. tdrtargets. org), a global open source database of molecular targets and drug like compounds that supports open innovation for infectious tropical diseases [38], [39].
The search for new drugs for human neglected diseases accelerated in the past decade, based on the recognition that addressing these infections was necessary for global poverty reduction. The expansion of discovery and development programmes was supported by donor investment, increasing participation of the industry and the creation of Product Development Partnership (PDP) enterprises. Despite these efforts, major discovery gaps remain as, apart from some repurposed drugs and a few new molecules for malaria, no new candidate has been recently transitioned from discovery into development for the major Neglected Tropical Diseases (NTDs). In this publication, we present a collaborative network model for drug discovery based on coordinated North-South partnerships. This network carried out low-to-medium throughput whole-organism screening assays against seven NTDs (malaria, leishmaniasis, human African trypanosomiasis [HAT], Chagas' disease, schistosomiasis, onchocerciasis and lymphatic filariasis) together with an early assessment of compound toxicity in mammalian cells. We describe a screening campaign of 10,000 molecules, its outcome and the implications of this strategy for enhancing the efficiency and productivity of drug discovery for NTDs.
toxicology
biochemistry
predictive toxicology
medicinal chemistry
small molecules
chemical biology
chemistry
biology
drug discovery
Integrated Dataset of Screening Hits against Multiple Neglected Disease Pathogens