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Trypanosomatid parasites cause devastating African sleeping sickness, Chagas disease, and Leishmaniasis that affect about 18 million people worldwide. Recently, we showed that the biogenesis of glycosomes could be the "Achilles' heel" of trypanosomatids suitable for the development of new therapies against trypanosomiases. This was shown for inhibitors of the import machinery of matrix proteins, while the distinct machinery for the topogenesis of glycosomal membrane proteins evaded investigation due to the lack of a druggable interface. Here we report on the identification of the highly divergent trypanosomal PEX3, a central component of the transport machinery of peroxisomal membrane proteins and the master regulator of peroxisome biogenesis. The trypanosomatid PEX3 shows very low degree of conservation and its identification was made possible by a combinatory approach identifying of PEX19-interacting proteins and secondary structure homology screening. The trypanosomal PEX3 localizes to glycosomes and directly interacts with the membrane protein import receptor PEX19. RNAi-studies revealed that the PEX3 is essential and that its depletion results in mislocalization of glycosomal proteins to the cytosol and a severe growth defect. Comparison of the parasites and human PEX3-PEX19 interface disclosed differences that might be accessible for drug development. The absolute requirement for biogenesis of glycosomes and its structural distinction from its human counterpart make PEX3 a prime drug target for the development of novel therapies against trypanosomiases. The identification paves the way for future drug development targeting PEX3, and for the analysis of additional partners involved in this crucial step of glycosome biogenesis.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5–PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5–PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3–PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3–TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3–TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.

African and American trypanosomiases are estimated to affect several million people across the world, with effective treatments distinctly lacking. New, ideally oral, treatments with higher efficacy against these diseases are desperately needed. Peroxisomal import matrix (PEX) proteins represent a very interesting target for structure- and ligand-based drug design. The PEX5-PEX14 protein-protein interface in particular has been highlighted as a target, with inhibitors shown to disrupt essential cell processes in trypanosomes, leading to cell death. In this work, we present a drug development campaign that utilizes the synergy between structural biology, computer-aided drug design, and medicinal chemistry in the quest to discover and develop new potential compounds to treat trypanosomiasis by targeting the PEX14-PEX5 interaction. Using the structure of the known lead compounds discovered by Dawidowski et al. as the template for a chemically advanced template search (CATS) algorithm, we performed scaffold-hopping to obtain a new class of compounds with trypanocidal activity, based on 2,3,4,5-tetrahydrobenzo[f][1,4]oxazepines chemistry. The initial compounds obtained were taken forward to a first round of hit-to-lead optimization by synthesis of derivatives, which show activities in the range of low- to high-digit micromolar IC50 in the in vitro tests. The NMR measurements confirm binding to PEX14 in solution, while immunofluorescent microscopy indicates disruption of protein import into the glycosomes, indicating that the PEX14-PEX5 protein-protein interface was successfully disrupted. These studies result in development of a novel scaffold for future lead optimization, while ADME testing gives an indication of further areas of improvement in the path from lead molecules toward a new drug active against trypanosomes. ISSN:1549-9596 ISSN:0095-2338 ISSN:1520-5142

The 7th Open European Peroxisome Meeting (OEPM) was held on the 15th and 16th of October 2020 due to the Corona pandemic as ZOOM conference. The OEPM is a biannual meeting organized by European expert researchers in peroxisome biology. Previous meetings were held in Leuven, Belgium (2006), Lunteren, The Netherlands (2010), Dijon, France (2012), Neuss, Germany (2014), Vienna, Austria (2016), and Groningen, The Netherlands (2018). For the 2020 meeting nearly 200 participants registered from 10 European countries, as well as Israel, Canada, the USA, Japan, Russia, and South Korea. At the OEPM, it is tradition that only young peroxisome researchers (PhD students or junior Post-docs) present their work to an international audience. At this year’s meeting, 46 talks were presented in eight sessions. During the meeting, the OEPM 2020 Peroxisome Research Young Investigator Award (sponsored by Ruhr-University of Bochum) was awarded. This award is given to the early career researcher, who was first author of the best publication in the peroxisome field over the last 2 years. At this meeting, the award was given to Victoria Riccio from the group of Peter Kim at the University of Toronto (Canada). The awarded paper describes that the deubiquitinating enzyme USP30 prevents pexophagy by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2. They also show that USP30 can rescue the peroxisome loss observed in some disease-causing peroxisome mutations, pointing to a potential therapeutic target (Riccio et al. 2019). Victoria Riccio received the prize during the meeting and presented the Young Investigator Award lecture about the paper. Members of the jury were Myriam Baes (Leuven), Mustapha Cherkaoui-Malki (Dijon), Ralf Erdmann (Bochum), Marc Fransen (Leuven), Aurora Pujol (Barcelona), Sigrun Reumann (Hamburg), Maya Schuldiner (Rehovot), Ron Wanders (Amsterdam), Bettina Warscheid (Freiburg), Hans Waterham (Amsterdam), and Einat Zalckvar (Rehovot). Iulia Revenco (KU Leuven, Belgium) and Marc Pilegaard Pedersen (University of Groningen, The Netherlands) presented the Marie Sklodowska-Curie Innovative European Training Network ‘PerICo’ (Peroxisome Interactions and Communication) that is coordinated by Prof. Dr. Ida van der Klei (University of Groningen). PerICo brings together ten full partners and five partner organizations, from seven European countries, together with Israel and Canada, and fosters the education of 15 PhD-students in projects aimed at uncovering how peroxisomes participate in cellular interactions and signaling. In the evening session, Andrew Longenecker presented ‘Diego’s story’, the case of his son Diego, who suffers from a PEX10 Zellweger Spectrum Disorder (ZSD), a peroxisome biogenesis disorder (PBD). In his presentation, he reported on a non-profit PBD-project (DiegoZSDResearch@gmail.com) that he and his wife created to engage with the research community and other parents. Peroxisomes are ubiquitous cell organelles of eukaryotic cells. They are surrounded by a single membrane and harbor a highly variable enzyme composition that determines the function of these multi-purpose organelles. Common functions of peroxisomes include β-oxidation of fatty acids and containment of pathways that produce hydrogen peroxide, which is then detoxified by catalase. Examples for species-specific peroxisomal pathways are alpha-oxidation of branched-chain fatty acids and synthesis of plasmalogens in humans, and contribution to the synthesis of penicillin in fungi or bile acids in humans (Wanders and Waterham 2006). Defects in peroxisomal enzymes or biogenesis of the organelle result in peroxisomal disorders, like X-linked Adrenoleukodystrophy or the Zellweger syndrome, respectively (Waterham et al. 2016). There is an increasing interest in peroxisome research, which is fueled by the fact that major questions concerning the biology of these fascinating organelles are still unanswered. Such as the import of folded, even oligomeric proteins into peroxisomes with the underlying mechanism still being unresolved (Baker et al. 2016; Walter and Erdmann 2019). Our knowledge on the biogenesis, dynamics (fission, transport), degradation by autophagy (pexophagy), etc. is still scarce, leaving room for exciting discoveries (Costello and Schrader 2018; Islinger et al. 2018; Mast et al. 2020; Mahalingam et al. 2020). Moreover, peroxisomes function in concert with other organelles, including metabolic interaction and signal transduction. In this context, an emerging theme is the investigation of physical contacts of peroxisomes and mitochondria, lipid droplets, vacuoles (lysosomes), and the ER via contact sites (Shai et al. 2016; Islinger et al. 2018; Farre et al. 2019; Schrader et al. 2020). Moreover, glycosomes, as peroxisomes of trypanosomatid parasites are called (due to the localization of glycolytic enzymes) have been identified as suitable targets for novel leads compounds for therapy of diseases like African sleeping sickness, Chagas Disease and Leishmaniasis (Dawidowski et al. 2017). Another reason for the steadily increasing interest is the perception that the cellular duties of these fascinating organelles go well beyond their undoubted metabolic functions. Peroxisomes turned out to have important non-metabolic roles, such as their function as signaling platform in antiviral response and thus their contribution to innate immunity (Ferreira et al. 2019), and their contribution to cooperative cell defense mechanisms against oxidative stress (Fujiki et al. 2020). Moreover, peroxisomes are critical contributors to ageing, longevity (Deori et al. 2018) and age-related disorders, including Alzheimer’s disease and cancer (Berger et al. 2016; Islinger et al. 2018; Dahabieh et al. 2018). Below is an overview of the sessions and talks of this very successful and inspiring OEPM. The order of talks at the meeting were essentially ordered alphabetically.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are ...

Vector-borne trypanosomatid parasite infections in tropical and sub-tropical countries constitute a major threat to humans and livestock. Trypanosoma brucei parasites are transmitted by tsetse fly and lead to African sleeping sickness in humans and Nagana in cattle. In Latin American countries, Trypanosoma cruzi infections spread by triatomine kissing bugs lead to Chagas disease. Various species of Leishmania transmitted to humans by phlebotomine sandflies manifest in a spectrum of diseases termed Leishmaniasis. 20 million people are currently infected with trypanosomatid parasites, leading to over 30,000 deaths annually and half billion people at risk of the infection. It is estimated that 300,000 Chagas infected people reside in the United States and 100,000 in Europe. Glycosomes are peroxisome-like organelles found only in trypanosomatids. Glycolysis occurs in the cytosol in all other organisms, but glycolytic enzymes and other metabolic pathways are compartmentalized inside glycosomes in trypanosomatids. Glycosomes are essential for the parasite survival and hence thought to be an attractive drug target. Our recent study [Dawidowski et al. Science (2017)] is the first to report small molecule inhibitors of glycosomal protein import. Using structure-based drug design, we developed small molecule inhibitors of the Trypanosoma PEX5-PEX14 protein-protein interaction that disrupt glycosomal protein import and kill the parasites. Oral treatment of T. brucei infected mice with PEX14 inhibitor significantly reduced the parasite levels with no adverse effect on mice. The study provides the grounds for further development of the glycosome inhibitors into clinical candidates and validates the parasite protein-protein interactions as drug targets.

The parasitic protists of the Trypanosoma genus infect humans and domestic mammals, causing severe mortality and huge economic losses. The most threatening trypanosomiasis is Chagas disease, affecting up to 12 million people in the Americas. We report a way to selectively kill Trypanosoma by blocking glycosomal/peroxisomal import that depends on the PEX14-PEX5 protein-protein interaction. We developed small molecules that efficiently disrupt the PEX14-PEX5 interaction. This results in mislocalization of glycosomal enzymes, causing metabolic catastrophe, and it kills the parasite. High-resolution x-ray structures and nuclear magnetic resonance data enabled the efficient design of inhibitors with trypanocidal activities comparable to approved medications. These results identify PEX14 as an "Achilles' heel" of the Trypanosoma suitable for the development of new therapies against trypanosomiases and provide the structural basis for their development.

Peroxisomes are dynamic organelles of eukaryotic cells performing a wide range of functions including fatty acid oxidation, peroxide detoxification and ether-lipid synthesis in mammals. Peroxisomes lack their own DNA and therefore have to import proteins post-translationally. Peroxisomes can import folded, co-factor bound and even oligomeric proteins. The involvement of cycling receptors is a special feature of peroxisomal protein import. Complex machineries of peroxin (PEX) proteins mediate peroxisomal matrix and membrane protein import. Identification of PEX genes was dominated by forward genetic techniques in the early 90s. However, recent developments in proteomic techniques has revolutionized the detailed characterization of peroxisomal protein import. Here, we summarize the current knowledge on peroxisomal protein import with emphasis on the contribution of proteomic approaches to our understanding of the composition and function of the peroxisomal protein import machineries.

Glycosomes evolved as specialized system for glycolysis in trypanosomatids. These organelle rely on protein import to maintain function. A machinery of peroxin (PEX) proteins is responsible for recognition and transport of glycosomal proteins to the organelle. Disruption of PEX-based import system was expected to be a strategy against trypanosomatids. Recently, a proof of this hypothesis has been presented. Here, we review current information about trypanosomatids' glycosomal transport components as targets for new trypanocidal therapies.

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

Peroxisomes are dynamic organelles of eukaryotic cells performing a wide range of functions including fatty acid oxidation, peroxide detoxification and ether-lipid synthesis in mammals. Peroxisomes lack their own DNA and therefore have to import proteins post-translationally. Peroxisomes can import folded, co-factor bound and even oligomeric proteins. The involvement of cycling receptors is a special feature of peroxisomal protein import. Complex machineries of peroxin (PEX) proteins mediate peroxisomal matrix and membrane protein import. Identification of PEX genes was dominated by forward genetic techniques in the early 90s. However, recent developments in proteomic techniques has revolutionized the detailed characterization of peroxisomal protein import. Here, we summarize the current knowledge on peroxisomal protein import with emphasis on the contribution of proteomic approaches to our understanding of the composition and function of the peroxisomal protein import machineries.

Protozoan parasites of the family Trypanosomatidae infect humans as well as livestock causing devastating diseases like sleeping sickness, Chagas disease, and Leishmaniasis. These parasites compartmentalize glycolytic enzymes within unique organelles, the glycosomes. Glycosomes represent a subclass of peroxisomes and they are essential for the parasite survival. Hence, disruption of glycosome biogenesis is an attractive drug target for these Neglected Tropical Diseases (NTDs). Peroxin 16 (PEX16) plays an essential role in peroxisomal membrane protein targeting and de novo biogenesis of peroxisomes from endoplasmic reticulum (ER). We identified trypanosomal PEX16 based on specific sequence characteristics and demonstrate that it is an integral glycosomal membrane protein of procyclic and bloodstream form trypanosomes. RNAi mediated partial knockdown of Trypanosoma brucei PEX16 in bloodstream form trypanosomes led to severe ATP depletion, motility defects and cell death. Microscopic and biochemical analysis revealed drastic reduction in glycosome number and mislocalization of the glycosomal matrix enzymes to the cytosol. Asymmetry of the localization of the remaining glycosomes was observed with a severe depletion in the posterior part. The results demonstrate that trypanosomal PEX16 is essential for glycosome biogenesis and thereby, provides a potential drug target for sleeping sickness and related diseases.

Peroxisomal biogenesis disorders (PBDs) represent a spectrum of autosomal recessive metabolic disorders that are collectively characterized by abnormal peroxisome assembly and impaired peroxisomal function. The importance of this ubiquitous organelle for human health is highlighted by the fact that PBDs are multisystemic disorders that often cause death in early infancy. Peroxisomes contribute to central metabolic pathways. Most enzymes in the peroxisomal matrix are linked to lipid metabolism and detoxification of reactive oxygen species. Proper assembly of peroxisomes and thus also import of their enzymes relies on specific peroxisomal biogenesis factors, so called peroxins with PEX being the gene acronym. To date, 13 PEX genes are known to cause PBDs when mutated. Studies of the cellular and molecular defects in cells derived from PBD patients have significantly contributed to the understanding of the functional role of the corresponding peroxins in peroxisome assembly. In this review, we discuss recent data derived from both human cell culture as well as model organisms like yeasts and present an overview on the molecular mechanism underlying peroxisomal biogenesis disorders with emphasis on disorders caused by defects in the peroxisomal matrix protein import machinery. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.