INTRODUCTION
The threat posed by anti-microbial resistance (AMR) has been well publicized with respect to bacterial pathogens, and the need to identify, validate and exploit new drug targets emphasized (Brown and Wright, Reference Brown and Wright2016). However, similar pressures exist for protozoan pathogens (Tuteja, Reference Tuteja2017; Uliana et al. Reference Uliana, Trinconi and Coelho2017). The Special Issue of Parasitology introduced here is focused on global infectious disease, namely the causative agents of the bacterial disease tuberculosis (TB; Mycobacterium tuberculosis) and the protozoal infections malaria, toxoplasmosis, leishmaniasis, African Sleeping Sickness and Chagas disease (the apicomplexans Plasmodium spp. and Toxoplasma gondii; and the kinetoplastids Leishmania spp., Trypanosoma brucei sp. and T. cruzi, respectively). Each of these poses serious challenges to health and wellbeing, and offer a multitude of challenges to effective treatment. Responsible for 1·8 m deaths, and with nearly 5% of the 10 million or more new cases each year showing multi-drug resistance, M. tuberculosis remains a serious problem, particularly in low- and middle-income countries (WHO, 2015). Similarly, although incidence levels fell more than 20% during 2010–2015, infection with mosquito-borne Plasmodium falciparum, the causative agent of serious malaria, remains a major global health problem leading to more than 200 m new cases and 400 000 deaths/year (WHO, 2016a ). The related apicomplexan protozoa Toxoplasma, classified by the Centers for Disease Control as causing a Neglected Parasitic Disease, chronically infects 30–60 m people in the USA alone, where it is considered a major food-borne pathogen (CDC, 2017). The kinetoplastid parasites Leishmania spp., T. cruzi and T. brucei sp. are insect-borne causes of Neglected Tropical Diseases (NTDs). However, whilst cases for African Sleeping Sickness caused by the later are declining [<3000 in 2015; (WHO, 2016b )], there are up to 1 m new cases of leishmaniasis per year, leading to 20 000 deaths and 6–7 m people remain infected with the parasite that causes Chagas disease (WHO, 2017). Indeed, the battle against Leishmania spp. and T. cruzi infection has recently been described as a losing one (Hotez and Aksoy, Reference Hotez and Aksoy2017).
For the global infections outlined above, the available drugs have limitations of efficacy, tolerance and/or administration, and cases of AMR are emerging or rampant. To address these problems, for both bacterial and protozoal pathogens there is a well recognized need to identify new targets for antimicrobial intervention (Brown and Wright, Reference Brown and Wright2016; Muller and Hemphill, Reference Muller and Hemphill2016). However, the identification, validation and understanding of new protein targets is not a straightforward process. For example, within the pharmaceutical industry there are wide-spread concerns regarding the reproducibility of drug target validation studies across a range of disease states (Jones, Reference Jones2016). Against this backdrop, the British Society for Parasitology 2016 Autumn Symposium focused on the identification, understanding and exploitation of targets for antibacterial and anti-protozoal intervention. In recognition of the industry concerns outlined above, the focus on cross-disciplinary analyses of putative targets was designed to answer the call to ‘embrace chemistry at the interface with biology’ (Jones, Reference Jones2016) and provide more robustly triaged drug targets. This approach necessitates the application of both state-of-the-art genetic and chemical tools to answer key questions in bioscience and robustly validate new drug targets in both bacterial and protozoan pathogens.
SEARCH FOR ANTIMICROBIAL TARGETS
With a crisis in antimicrobial resistance upon us, and the persistence of neglected infectious diseases (e.g. NTDs), new drug leads need to be rapidly identified. High-throughput screening (HTS) remains at the centre of drug discovery and can be carried out using either in vitro assays against validated targets or phenotypic assays against the pathogen itself (Denny and Steel, Reference Denny and Steel2014; Norcliffe et al. Reference Norcliffe, Alvarez-Ruiz, Martin-Plaza, Steel and Denny2014). Recent high content phenotypic screening across the kinetoplastids gave a disappointingly low number of novel potent hits against Leishmania donovani when compared with the related parasite T. brucei (Pena et al. Reference Pena, Manzano, Cantizani, Kessler, Alonso-Padilla, Bardera, Alvarez, Rodriquez, Gray, Navarro, Kumar, Sherstnev, Drewry, Brown, Fiandor and Julio Martin2015). Phenotypic HTS has been successfully carried out against M. tuberculosis, for example using genetically modified bacteria in a resistance based screen (Cox et al. Reference Cox, Mugumbate, Del Peral, Jankute, Abrahams, Jervis, Jackenkroll, Perez, Alemparte, Esquivias, Lelièvre, Ramon, Barros, Ballell and Besra2016). However, as for the kinetoplastids this has proven problematic, due in large part to the slow growth rate of M. tuberculosis (White et al. Reference White, Tower and Rasmussen2016). These studies demonstrated that target-based screening remains vital of for antimicrobial discovery and, of course, this relies upon the provision of high quality, fully validated, antimicrobial drug targets.
Natural product antibacterials (antibiotics) targeting the cell wall have long been in clinical use, many of these are directed against peptidoglycan, the principle component of Gram-positive and -negative bacterial walls (Muller et al. Reference Muller, Klockner and Schneider2017). Such antimicrobials show excellent selectivity for the synthesis of this non-mammalian structure; however, they are at the forefront of concerns regarding AMR. Likewise, several anti-TB agents target cell wall synthesis, however the M. tuberculosis wall has several unique features, which present challenges for the development new chemotherapeutics. For example, the long-chain mycolic acids which cover the cell surface facilitate the intercalation of acyl lipids forming a waxy outer membrane, which forms a hydrophobic barrier. Despite these obstacles the M. tuberculosis wall and its biosynthesis remains an important and attractive target for novel anti-TB drugs, as concluded in the first review in this Special Issue (Abrahams and Besra, Reference Abrahams and Besra2016). Abrahams and Besra present the biosynthesis of this essential structural and permeability barrier as being the ‘Achilles heel’ of this pathogen and open up the prospect of modern approaches to drug discovery (e.g. HTS) identifying novel therapeutics.
Similarly, the protozoan sphingolipid biosynthetic pathway has been proposed as a possible drug target for kinetoplastid (e.g. Leishmania spp. and T. brucei) and apicomplexan (Plasmodium spp. and Toxoplasma) eukaryotic pathogens (Mina et al. Reference Mina, Mosely, Ali, Shams-Eldin, Schwarz, Steel and Denny2010, Reference Mina, Mosely, Ali, Denny and Steel2011; Young et al. Reference Young, Mina, Denny and Smith2012; Coppens, Reference Coppens2013; Pratt et al. Reference Pratt, Wansadhipathi-Kannangara, Bruce, Mina, Shams-Eldin, Casas, Hanada, Schwarz, Sonda and Denny2013). Against the backdrop of ancient, toxic therapies and rising AMR (Barrett and Croft, Reference Barrett and Croft2014) the essentiality of sphingolipids, and the potential to target their biosynthesis, has seen growing interest. In a ‘state-of-the-art’ review in this Special Issue, Mina and Denny discuss possibilities and pitfalls of targeting this biosynthetic pathway, considering both parasite de novo synthesis and host scavenging (Mina and Denny, Reference Mina and Denny2017). Key differences between the mammalian host sphingolipid biosynthetic pathway and that of both kinetoplastid (Denny et al. Reference Denny, Shams-Eldin, Price, Smith and Schwarz2006; Zhang et al. Reference Zhang, Bangs and Beverley2010) and apicomplexan (Coppens, Reference Coppens2013; Pratt et al. Reference Pratt, Wansadhipathi-Kannangara, Bruce, Mina, Shams-Eldin, Casas, Hanada, Schwarz, Sonda and Denny2013; Mina and Denny, Reference Mina and Denny2017) protozoan parasites, have fuelled this endeavour. In a companion piece, Alqaisi et al. (Reference Alqaisi, Mbekeani, Llorens, Elhammer and Denny2017) describe an investigation of the mode of action of a reported inhibitor of Toxoplasma sphingolipid biosynthesis, aureobasidin A. However, whilst this natural compound is antiparasitic against both acute and chronic forms, parasite sphingolipid biosynthesis was unaffected.
Remaining in the field of lipid biochemistry, protein acylation has long been proposed as a target of novel antiprotozoals, with the enzyme responsible for the essential N-myristoylation of proteins [N-myristoyl transferase (NMT)] identified as a potential drug target in apicomplexan (Gunaratne et al. Reference Gunaratne, Sajid, Ling, Tripathi, Pachebat and Holder2000) and kinetoplastid (Price et al. Reference Price, Menon, Panethymitaki, Goulding, McKean and Smith2003) protozoan parasites. In this Issue, the use of chemical proteomic approaches to analyse and validate such post-translation modifications is discussed, with reference to both N-myristoylation and S-palmitoylation (Ritzefeld et al. Reference Ritzefeld, Wright and Tate2017). The use of chemical tools (such as acyl biotin exchange and metabolic tagging with click chemistry) is essential to fully understand the downstream effects of enzyme inhibition and provide further validation of targets and inhibitors (Tate et al. Reference Tate, Bell, Rackham and Wright2014; Ritzefeld et al. Reference Ritzefeld, Wright and Tate2017).
A unique target in kinetoplastid protozoa is the mitochondrial protein, trypanosome alternative oxidase (TAO). This target is now well characterized in T. brucei where it has ubiquinol oxidase activity and is expressed more than 100-fold more in pathogenic bloodstream forms (Chaudhuri et al. Reference Chaudhuri, Ajayi and Hill1998). Functionally it is thought to protect the parasite from oxidative damage (Fang and Beattie, Reference Fang and Beattie2003). As reviewed in this issue, inhibitors of TAO have been identified which are able to clear infection in vivo (Menzies et al. Reference Menzies, Tulloch, Florence and Smith2016).
Collectively, these studies and associated reviews emphasize the place for a target-directed approach in antimicrobial discover and emphasize the importance of chemical approaches for the understanding and validation of drug targets.
EXPLORATION AND EXPLOITATION OF ANTIMICROBIAL TARGETS
As discussed above, screening of phenotypic changes in response to chemical assault is one approach to identifying new leads as antiparasitics, although obtaining informative readouts from such assays can be complex (Denny and Steel, Reference Denny and Steel2014; Norcliffe et al. Reference Norcliffe, Alvarez-Ruiz, Martin-Plaza, Steel and Denny2014). In this Special Issue, the use of in silico synchronization, using defined cell parameters, to more readily analyse the cell cycle of T. brucei is proposed (Morriswood and Engstler, Reference Morriswood and Engstler2017). Such an automated process could increase throughput and standardize data quantitation, perhaps providing more robust phenotypic data. However, if an antimicrobial target is in hand, the search for inhibitors for use as drug leads or chemical tools for further validation and understanding, can employ conventional HTS. Typically, this involves screening a large, diverse compound library (>100 k) against a protein target in a multiwell formatted biochemical assay (Denny and Steel, Reference Denny and Steel2014; Norcliffe et al. Reference Norcliffe, Alvarez-Ruiz, Martin-Plaza, Steel and Denny2014). However, fragment-based approaches are now often run alongside such HTS, and have been the key to success in several drug discovery programmes (Congreve et al. Reference Congreve, Chessari, Tisi and Woodhead2008; Scott et al. Reference Scott, Coyne, Hudson and Abell2012). The application of this approach in the discovery of inhibitors of M. tuberculosis targets is reviewed here by Marchetti et al. A fragment-based approach involves screening a small library (1000–5000) of fragments (<250 Da) against a protein target, and identifying weak binders by using a variety of biophysical tools such as surface plasmon resonance and nuclear magnetic resonance (Marchetti et al. Reference Marchetti, Chan, Coyne and Abell2016). Notable successes include the identification, using thermal shift assays, of fragments binding to EthR, a TetR-type transcriptional repressor that underlies M. tuberculosis resistance to the second-line drug ethionamide (Villemagne et al. Reference Villemagne, Flipo, Blondiaux, Crauste, Malaquin, Leroux, Piveteau, Villeret, Brodin, Villoutreix, Sperandio, Wohlkönig, Wintjens, Deprez, Baulard and Willand2014). Following analyses of an X-ray co-crystal structure, a virtual library was designed and screened in silico, leading to the identification of derivatives with high in vitro activity (Tatum et al. Reference Tatum, Villemagne, Willand, Deprez, Liebeschuetz, Baulard and Pohl2013; Villemagne et al. Reference Villemagne, Flipo, Blondiaux, Crauste, Malaquin, Leroux, Piveteau, Villeret, Brodin, Villoutreix, Sperandio, Wohlkönig, Wintjens, Deprez, Baulard and Willand2014). This demonstrates the centrality of high resolution protein structures to fragment-based ligand-discovery approaches (Murray and Blundell, Reference Murray and Blundell2010), and the potential of in silico screening.
Such structure-based approaches can be considered as applicable to all protein targets, in this Issue the calcium-dependent protein kinases (CDPK) from Toxoplasma are considered as targets for such an approach (Cardew et al. Reference Cardew, Verlinde and Pohl2017). CDPK are restricted to plants and protozoa and have been genetically demonstrated to be essential in multiple systems, including Toxoplasma, presenting them as attractive drug targets (Long et al. Reference Long, Wang and Sibley2016; Wang et al. Reference Wang, Huang, Li, Chen, Ning and Zhu2016). Structure-based approaches have led to the discovery of potent CDPK inhibitors, with specificity with respect to mammalian kinases and good antiparasitic activity (Lourido et al. Reference Lourido, Jeschke, Turk and Sibley2013; Zhang et al. Reference Zhang, Ojo, Vidadala, Huang, Geiger, Scheele, Choi, Reid, Keyloun, Rivas, Siddaramaiah, Comess, Robinson, Merta, Kifle, Hol, Parsons, Merritt, Maly, Verlinde, Van Voorhis and Fan2014; Moine et al. Reference Moine, Dimier-Poisson, Enguehard-Gueiffier, Loge, Penichon, Moire, Delehouze, Foll-Josselin, Ruchaud, Bach, Gueiffier, Debierre-Grockiego and Denevault-Sabourin2015).
This work, taken together, illustrates the power of utilising diverse chemical and biophysical approaches to identify novel inhibitors and antimicrobial lead compounds.
ALTERNATIVE APPROACHES AND DOWNSTREAM NECESSITY
Whilst target-based approaches to antimicrobial discovery remain central, the exceedingly long history of repurposing drugs for use as antiparasitics demonstrates that we should not be too narrow in our thinking. As reviewed in this Special Issue repurposing has the potential to significantly reduce the costs of antimicrobial discovery by bypassing the initial development phases necessary for new chemical entities (Charlton et al. Reference Charlton, Rossi-Bergmann, Denny and Steel2017). Charlton et al. discuss the current prominence of such drugs in the treatment of leishmaniasis, for example amphotericin B, which was developed as an antifungal but is currently in use in the South Asian visceral leishmaniais elimination programme (Gurunath et al. Reference Gurunath, Joshi, Agrawal and Shah2014). In addition, they review the history and potential of other drugs developed as, for example, antiviral and anticancer agents, for use as antileishmanials. However, as Charlton et al. recognize, whilst the discovery and repurposing of existing pharmaceuticals will save time and money in the vital search for safe, effective and affordable antileishmanials, the identification of the mode of action of such drugs is essential of further development (Ritzefeld et al. Reference Ritzefeld, Wright and Tate2017).
The discovery and validation of antimicrobial targets and potent inhibitors is, of course, a vital component of a drug discovery programme. However, these in vitro approaches provide no indication as to the ability of identified chemical entities to reach the target pathogen within the host. The particularly acute challenges of this stage in the discovery pipeline for pathogens sequestered within host cells, such as Leishmania spp. and M. tuberculosis, is reviewed in this Issue (Croft, Reference Croft2017). The integration of pharmacokinetics (PK), pharmacodynamics (PD) and physiological-modelling into the antimicrobial drug discovery process has been previously reviewed (Edginton et al. Reference Edginton, Theil, Schmitt and Willmann2008; Nielsen and Friberg, Reference Nielsen and Friberg2013), and the importance of PK–PD analyses in M. tuberculosis drug design demonstrated (Davies and Nuermberger, Reference Davies and Nuermberger2008; Dartois, Reference Dartois2014). Given that Leishmania spp. occupy a similar intracellular site to M. tuberculosis, Croft considers the application of these approaches to antileishmanial discovery, concluding that uniform approaches at all levels of the pipeline are vital to ensure the development process can proceed as rapidly as possible.
Concluding remarks
The collection within this Special Issue illustrates the centrality of high quality target validation (using both biological and physical methodologies); the importance of multifaceted inhibitor discovery (integrating HTS and biophysical approaches); and the requirement to consider physiological factors (such as PK–PD) in antimicrobial discovery. In summary, the adoption of the multidisciplinary approaches outlined is essential to accelerate the discovery of new drugs to treat the most prevalent, and often intractable, global infections caused by both bacterial and protozoal pathogens.
FINANCIAL SUPPORT
The British Society for Parasitology and I would like to thank the Royal Society of Chemistry, GSK, Durham University Wolfson and Biophysical Research Institutes, and Cambridge University Press for sponsorship of the 2016 Autumn Symposium held at Durham University. PWD is supported by grants from the Medical Research Council (MR/P027989/1) and Biotechnology and Biological Research Council (BB/M024156/1 and NPRONET).
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