Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 in apicomplexan parasites with a glycine gatekeeper, are promising new therapeutics for apicomplexan diseases. Here we will review advances, as well as challenges and lessons learned regarding efficacy, safety, and pharmacology that have shaped our selection of pre-clinical candidates.

Small Molecule Inhibitors Targeting the Heat Shock Protein System of Human Obligate Protozoan Parasites.

Obligate protozoan parasites of the kinetoplastids and apicomplexa infect human cells to complete their life cycles. Some of the members of these groups of parasites develop in at least two systems, the human host and the insect vector. Survival under the varied physiological conditions associated with the human host and in the arthropod vectors requires the parasites to modulate their metabolic complement in order to meet the prevailing conditions. One of the key features of these parasites essential for their survival and host infectivity is timely expression of various proteins. Even more importantly is the need to keep their proteome functional by maintaining its functional capabilities in the wake of physiological changes and host immune responses. For this reason, molecular chaperones (also called heat shock proteins)-whose role is to facilitate proteostasis-play an important role in the survival of these parasites. Heat shock protein 90 (Hsp90) and Hsp70 are prominent molecular chaperones that are generally induced in response to physiological stress. Both Hsp90 and Hsp70 members are functionally regulated by nucleotides. In addition, Hsp70 and Hsp90 cooperate to facilitate folding of some key proteins implicated in cellular development. In addition, Hsp90 and Hsp70 individually interact with other accessory proteins (co-chaperones) that regulate their functions. The dependency of these proteins on nucleotide for their chaperone function presents an Achille's heel, as inhibitors that mimic ATP are amongst potential therapeutic agents targeting their function in obligate intracellular human parasites. Most of the promising small molecule inhibitors of parasitic heat shock proteins are either antibiotics or anticancer agents, whose repurposing against parasitic infections holds prospects. Both cancer cells and obligate human parasites depend upon a robust protein quality control system to ensure their survival, and hence, both employ a competent heat shock machinery to this end. Furthermore, some inhibitors that target chaperone and co-chaperone networks also offer promising prospects as antiparasitic agents. The current review highlights the progress made so far in design and application of small molecule inhibitors against obligate intracellular human parasites of the kinetoplastida and apicomplexan kingdoms.

Same same, but different: Uncovering unique features of the mitochondrial respiratory chain of apicomplexans.

Mitochondrial respiration is a critical process for the survival of many eukaryotes, including parasites in the phylum Apicomplexa. These intracellular parasites include the causative agents of numerous serious diseases in humans and animals, including toxoplasmosis (Toxoplasma gondii) and malaria (Plasmodium species). Emerging evidence indicates that the mitochondrial respiratory chain of apicomplexans has notable differences to that of the host cells they infect. These differences make the respiratory chain a prominent drug target in apicomplexans, with numerous inhibitors of this pathway in current use or development. This review highlights unique aspects of the respiratory chain of apicomplexans and provides perspective on emerging points of inquiry into this essential and therapeutically exploitable pathway.

Druggable Targets in Cyclic Nucleotide Signaling Pathways in Apicomplexan Parasites and Kinetoplastids against Disabling Protozoan Diseases in Humans.

Cell signaling in eukaryotes is an evolutionarily conserved mechanism to respond and adapt to various environmental changes. In general, signal sensation is mediated by a receptor which transfers the signal to a cascade of effector proteins. The cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers mediating an extracellular stimulus to cyclic nucleotide-dependent kinases driving a change in cell function. In apicomplexan parasites and kinetoplastids, which are responsible for a variety of neglected, tropical diseases, unique mechanisms of cyclic nucleotide signaling are currently identified. Collectively, cyclic nucleotides seem to be essential for parasitic proliferation and differentiation. However, there is no a genomic evidence for canonical G-proteins in these parasites while small GTPases and secondary effector proteins with structural differences to host orthologues occur. Database entries encoding G-protein-coupled receptors (GPCRs) are still without functional proof. Instead, signals from the parasite trigger GPCR-mediated signaling in the host during parasite invasion and egress. The role of cyclic nucleotide signaling in the absence of G-proteins and GPCRs, with a particular focus on small GTPases in pathogenesis, is reviewed here. Due to the absence of G-proteins, apicomplexan parasites and kinetoplastids may use small GTPases or their secondary effector proteins and host canonical G-proteins during infection. Thus, the feasibility of targeting cyclic nucleotide signaling pathways in these parasites, will be an enormous challenge for the identification of selective, pharmacological inhibitors since canonical host proteins also contribute to pathogenesis.

Puromycin selection for stable transfectants of the oyster-infecting parasite Perkinsus marinus.

Perkinsus marinus is a marine protozoan parasite that infects natural and farmed oysters, attracting attention from researchers in both fisheries and evolutionary biology. The functions of almost all cellular components and organelles are, however, poorly understood even though a draft genome sequence of P. marinus is publicly available. One of the major obstacles for a functional study of the parasite is limited experimental means for genetic manipulation: a transfection method was established in 2008, and the first drug selection system with bleomycin was reported in 2016. We here introduce the second drug-selectable marker for selection of P. marinus transfectants. The parasite growth is efficiently inhibited by puromycin (IC50 = 4.96 µg/mL), and transfection of its resistance gene, puromycin-N-acetyl-transferase (pac), confers resistance to the drug on the parasite. Stable transfectants can be obtained within 2 months by treating with puromycin at 100 µg/mL. Furthermore, combining puromycin and bleomycin treatment can select transfectants co-expressing two marker genes. This dual-transfection method raises the possibility of using co-localization to identify the cellular localization of novel proteins in P. marinus, thereby contributing to the understanding of cellular functions and pathogenesis.

Effect of jasplakinolide and cytochalasin D on cortical elements involved in the gliding motility of the eugregarine Gregarina garnhami (Apicomplexa).

Since apicomplexans represent exclusively parasitic unicellular organisms with medical and economic impacts, the principles of their motility have been studied intensively. By contrast, the movement in apicomplexan basal groups, such as gregarines, remains to be elucidated. The present study focuses on Gregarina garnhami parasitising the digestive tract of the locust Schistocerca gregaria, and investigates the involvement of cytoskeletal elements (the ectoplasmic network and myonemes) and the secretion of mucosubstances during eugregarine gliding motility. Combined microscopic analyses were used to verify the role of actin filaments and membranes' organisation in G. garnhami motility. A freeze-etching analysis of membranes revealed the size, density, and arrangement of intramembranous particles along with the distribution and size of pores and ducts. Experimental assays using actin-modifying drugs (jasplakinolide, cytochalasin D) confirmed that actin most likely plays a role in cell motility, principally in its filamentous form (=F-actin). Myonemes, localised in the border between the ectoplasm and endoplasm, correspond to the concentric bundles of F-actin. Microscopic analyses confirmed that changes in gamonts motility corresponding to the changes in the organisation and density of myonemes and the ectoplasmic network in drug-treated cells, suggesting that these structures might serve as contractile elements facilitating gliding motility in G. garnhami.

A minimalistic approach to develop new anti-apicomplexa polyamines analogs.

The development of new chemical entities against the major diseases caused by parasites is highly desired. A library of thirty diamines analogs following a minimalist approach and supported by chemoinformatics tools have been prepared and evaluated against apicomplexan parasites. Different member of the series of N,N'-disubstituted aliphatic diamines shown in vitro activities at submicromolar concentrations and high levels of selectivity against Toxoplasma gondii and in chloroquine-sensitive and resistant-strains of Plasmodium falciparum. In order to demonstrate the importance of the secondary amines, ten N,N,N',N'-tetrasubstituted aliphatic diamines derivatives were synthesized being considerably less active than their disubstituted counterpart. Theoretical studies were performed to establish the electronic factors that govern the activity of the compounds.

Everybody needs sphingolipids, right! Mining for new drug targets in protozoan sphingolipid biosynthesis.

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the de novo synthesis or the degradation pathways has been shown to have detrimental effects. The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.

Motility in blastogregarines (Apicomplexa): Native and drug-induced organisation of Siedleckia nematoides cytoskeletal elements.

Recent studies on motility of Apicomplexa concur with the so-called glideosome concept applied for apicomplexan zoites, describing a unique mechanism of substrate-dependent gliding motility facilitated by a conserved form of actomyosin motor and subpellicular microtubules. In contrast, the gregarines and blastogregarines exhibit different modes and mechanisms of motility, correlating with diverse modifications of their cortex. This study focuses on the motility and cytoskeleton of the blastogregarine Siedleckia nematoides Caullery et Mesnil, 1898 parasitising the polychaete Scoloplos cf. armiger (Müller, 1776). The blastogregarine moves independently on a solid substrate without any signs of gliding motility; the motility in a liquid environment (in both the attached and detached forms) rather resembles a sequence of pendular, twisting, undulation, and sometimes spasmodic movements. Despite the presence of key glideosome components such as pellicle consisting of the plasma membrane and the inner membrane complex, actin, myosin, subpellicular microtubules, micronemes and glycocalyx layer, the motility mechanism of S. nematoides differs from the glideosome machinery. Nevertheless, experimental assays using cytoskeletal probes proved that the polymerised forms of actin and tubulin play an essential role in the S. nematoides movement. Similar to Selenidium archigregarines, the subpellicular microtubules organised in several layers seem to be the leading motor structures in blastogregarine motility. The majority of the detected actin was stabilised in a polymerised form and appeared to be located beneath the inner membrane complex. The experimental data suggest the subpellicular microtubules to be associated with filamentous structures (= cross-linking protein complexes), presumably of actin nature.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Investigation into the Physiological Significance of the Phytohormone Abscisic Acid in Perkinsus marinus, an Oyster Parasite Harboring a Nonphotosynthetic Plastid.

Some organisms have retained plastids even after they have lost the ability to photosynthesize. Several studies of nonphotosynthetic plastids in apicomplexan parasites have shown that the isopentenyl pyrophosphate biosynthesis pathway in the organelle is essential for their survival. A phytohormone, abscisic acid, one of several compounds biosynthesized from isopentenyl pyrophosphate, regulates the parasite cell cycle. Thus, it is possible that the phytohormone is universally crucial, even in nonphotosynthetic plastids. Here, we examined this possibility using the oyster parasite Perkinsus marinus, which is a plastid-harboring cousin of apicomplexan parasites and has independently lost photosynthetic ability. Fluridone, an inhibitor of abscisic acid biosynthesis, blocked parasite growth and induced cell clustering. Nevertheless, abscisic acid and its intermediate carotenoids did not affect parasite growth or rescue the parasite from inhibition. Moreover, abscisic acid was not detected from the parasite using liquid chromatography mass spectrometry. Our findings show that abscisic acid does not play any significant roles in P. marinus.

Targeting Protein Translation in Organelles of the Apicomplexa.

Antibiotics inhibiting protein translation have long been used to treat and prevent infections by apicomplexan parasites. These compounds kill parasites by inhibiting organellar translation, and most act specifically against the apicoplast, a relict plastid in apicomplexans. Drug resistance in Plasmodium and other apicomplexans dictates a need for development of novel targets. Some apicoplast inhibitors have a delayed onset of action, so they cannot replace fast-acting drugs, although they still fulfil important roles in treating and preventing infections. The plethora of bacterial-like actors in the translation machinery of parasite mitochondria and plastids presents validated targets with strong potential for selectivity. Here we discuss existing drugs that inhibit organellar translation, and explore targets that may be further exploited in antiparasitic drug design.

A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity.

An in vitro screening of the anti-apicomplexan activity of 51 compounds, stemming from our chemical library and from chemical synthesis, was performed. As a study model, we used Toxoplasma gondii (T. gondii), expressing ß-galactosidase for the colorimetric assessment of drug activity on parasites cultivated in vitro. This approach allowed the validation of a new series of molecules with a biphenylimidazoazine scaffold as inhibitors of T. gondii growth in vitro. Hence, 8 molecules significantly inhibited intracellular replication of T. gondii in vitro, with EC50 < 1 µM, while being non-toxic for human fibroblasts at these concentrations. Most attractive candidates were then selected for further biological investigations on other apicomplexan parasites (Neospora caninum, Besnoitia besnoiti, Eimeria tenella and Plasmodium falciparum). Finally, two compounds were able to inhibit growth of four different apicomplexans with EC50 in the submicromolar to nanomolar range, for each parasite. These data, including the broad anti-parasite spectrum of these inhibitors, define a new generation of potential anti-parasite compounds of wide interest, including for veterinary application. Studies realized on E. tenella suggest that these molecules act during the intracellular development steps of the parasite. Further experiments should be done to identify the molecular target(s) of these compounds.

Chemical biology approaches for the study of apicomplexan parasites.

Chemical biology and the techniques the field encompasses provide scientists with the means to address biological questions in ever-evolving and technically sophisticated ways. They facilitate the dissection of molecular mechanisms of cell phenomena on timescales not achievable by other means. Libraries of small molecules, bioorthogonal chemistries and technical advances in mass-spectrometry techniques enable the modern chemical biologist to tackle even the most difficult of biological questions. It is because of their broad applicability that these approaches are well suited to systems less tractable to more classical genetic methods. As such, the parasite community has embraced them with great success. Some of these successes and the continuing evolution of chemical biology applied to apicomplexans will be discussed.

The shikimate pathway in apicomplexan parasites: implications for drug development.

The shikimate pathway provides basic building blocks for a variety of aromatic compounds including aromatic amino acids, ubiquinone, folate and compounds of the secondary metabolism. The seven enzymatic reactions of the pathway lead to the generation of chorismate from simple products of the carbohydrate metabolism, namely erythrose 4-phosphate and phosphoenolpyruvate. The shikimate pathway is present in plants, bacteria, fungi and chromalveolata to which the apicomplexan parasites belong. As it is absent from humans, the enzymes of the shikimate pathway are attractive targets for antimicrobial drug development. Inhibition of the pathway is effective in controlling growth of certain apicomplexan parasites including the malaria parasite Plasmodium falciparum. Yet, despite being an attractive drug target, our knowledge of the shikimate pathway in this parasite group is lacking. The current review summarizes the available information and discusses aspects of the genetic organization of the shikimate pathway in apicomplexan parasites. Compounds acting on shikimate pathway enzymes will be presented and discussed in light of their impact for antiapicomplexan/antiplasmodial drug development.

In vitro culture systems for the study of apicomplexan parasites in farm animals.

In vitro culture systems represent powerful tools for the study of apicomplexan parasites such as Cryptosporidium, Eimeria, Sarcocystis, Neospora, Toxoplasma, Besnoitia, Babesia and Theileria, all with high relevance for farm animals. Proliferative stages of these parasites have been cultured in vitro employing a large variety of cell culture and explant approaches. For some, such as Cryptosporidium and Eimeria, the sexual development has been reproduced in cell cultures, while for others, animal experimentation is required to fulfill the life cycle. In vitro cultures have paved the way to exploit the basic biology of these organisms, and had a major impact on the development of tools for diagnostic purposes. With the aid of in vitro cultivation, studies on host-parasite interactions, on factors involved in innate resistance, stage conversion and differentiation, genetics and transfection technology, vaccine candidates and drug effectiveness could be carried out. The use of transgenic parasites has facilitated high-throughput screening of anti-microbial compounds that are active against the proliferative stages. Here, we review the basic features of cell culture-based in vitro systems for apicomplexan parasites that are relevant for farm animals, and discuss their applications with a focus on drug identification and studies of stage differentiation.

Autophagy in Apicomplexa: a life sustaining death mechanism?

Programmed cell death (PCD) pathways remain understudied in parasitic protozoa in spite of the fact that they provide potential targets for the development of new therapy. The best understood PCD pathway in higher eukaryotes is apoptosis although emerging evidence also points to autophagy as a mediator of death in certain physiological contexts. Bioinformatic analyses coupled with biochemical and cell biological studies suggest that parasitic protozoa possess the capacity for PCD including a primordial form of apoptosis. Recent work in Toxoplasma and emerging data from Plasmodium suggest that autophagy-related processes may serve as an additional death promoting pathway in Apicomplexa. Detailed mechanistic studies into the molecular basis for PCD in parasitic protozoa represent a fertile area for investigation and drug development.

Metronidazole induces gametocytogenesis in gregarine associations maintained in vitro.

Gametocytogenesis was induced in mature associations of Protomagalhaensia wolfi and Protomagalhaensia blaberae maintained in vitro by inclusion of metronidazole in the culture medium. The response was neither strictly dosage dependent nor uniform across gregarine species. We hypothesize that metronidazole induces gregarine gametocytogenesis by disrupting PUF2 proteins responsible for the translational control of sexual development and gametocytogenesis in apicomplexans.

Roles of Apicomplexan protein kinases at each life cycle stage.

Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma.

Ribonucleotide reductase as a target to control apicomplexan diseases.

Malaria is caused by species in the apicomplexan genus Plasmodium, which infect hundreds of millions of people each year and kill close to one million. While malaria is the most notorious of the apicomplexan-caused diseases, other members of eukaryotic phylum Apicomplexa are responsible for additional, albeit less well-known, diseases in humans, economically important livestock, and a variety of other vertebrates. Diseases such as babesiosis (hemolytic anemia), theileriosis and East Coast Fever, cryptosporidiosis, and toxoplasmosis are caused by the apicomplexans Babesia, Theileria, Cryptosporidium and Toxoplasma, respectively. In addition to the loss of human life, these diseases are responsible for losses of billions of dollars annually. Hence, the research into new drug targets remains a high priority. Ribonucleotide reductase (RNR) is an essential enzyme found in all domains of life. It is the only means by which de novo synthesis of deoxyribonucleotides occurs, without which DNA replication and repair cannot proceed. RNR has long been the target of antiviral, antibacterial and anti-cancer therapeutics. Herein, we review the chemotherapeutic methods used to inhibit RNR, with particular emphasis on the role of RNR inhibition in Apicomplexa, and in light of the novel RNR R2_e2 subunit recently identified in apicomplexan parasites.