Filarial DAF-12 sense the host serum to resume iL3 development during infection.

Nematode parasites enter their definitive host at the developmentally arrested infectious larval stage (iL3), and the ligand-dependent nuclear receptor DAF-12 contributes to trigger their development to adulthood. Here, we characterized DAF-12 from the filarial nematodes Brugia malayi and Dirofilaria immitis and compared them with DAF-12 from the non-filarial nematodes Haemonchus contortus and Caenorhabditis elegans. Interestingly, Dim and BmaDAF-12 exhibit high sequence identity and share a striking higher sensitivity than Hco and CelDAF-12 to the natural ligands Δ4- and Δ7-dafachronic acids (DA). Moreover, sera from different mammalian species activated specifically Dim and BmaDAF-12 while the hormone-depleted sera failed to activate the filarial DAF-12. Accordingly, hormone-depleted serum delayed the commencement of development of D. immitis iL3 in vitro. Consistent with these observations, we show that spiking mouse charcoal stripped-serum with Δ4-DA at the concentration measured in normal mouse serum restores its capacity to activate DimDAF-12. This indicates that DA present in mammalian serum participate in filarial DAF-12 activation. Finally, analysis of publicly available RNA sequencing data from B. malayi showed that, at the time of infection, putative gene homologs of the DA synthesis pathways are coincidently downregulated. Altogether, our data suggest that filarial DAF-12 have evolved to specifically sense and survive in a host environment, which provides favorable conditions to quickly resume larval development. This work sheds new light on the regulation of filarial nematodes development while entering their definitive mammalian host and may open the route to novel therapies to treat filarial infections.

Anticoccidial drugs of the livestock industry.

Coccidiosis is a parasitic disease of a wide variety of animals caused by coccidian protozoa. The coccidia are responsible for major economic losses of the livestock industry. For example, the annual cost due to coccidiosis to the global poultry industry has been estimated to exceed US$ 3 billion annually. Currently available drugs for the control of this disease are either polyether ionophorous antibiotics that are derived from fermentation products, or synthetic compounds, produced by chemical synthesis. Unfortunately, no new drugs in either category have been approved for use for decades. Resistance has been documented for all those of the drugs currently employed and therefore the discovery of novel drugs with unique modes of action is imperative if chemotherapy is to remain the principal means to control this disease. This chapter aims to give an overview of the efficacy and mode of action of the current compounds used to control coccidiosis in livestock and provides a brief outlook of research needs for the future.

Antimicrobial Preservatives for Protein and Peptide Formulations: An Overview.

Biological drugs intended for multi-dose application require the presence of antimicrobial preservatives to avoid microbial growth. As the presence of certain preservatives has been reported to increase protein and peptide particle formation, it is essential to choose a preservative compatible with the active pharmaceutical ingredient in addition to its preservation function. Thus, this review describes the current status of the use of antimicrobial preservatives in biologic formulations considering (i) appropriate preservatives for protein and peptide formulations, (ii) their physico-chemical properties, (iii) their in-/compatibilities with other excipients or packaging material, and (iv) their interactions with the biological compound. Further, (v) we present an overview of licensed protein and peptide formulations.

Identification of lead compounds targeting the cathepsin B-like enzyme of Eimeria tenella.

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a.

The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.

Antiparasitics in Animal Health: Quo Vadis?

Antiparasitics acting on endo- or ectoparasites represent the second largest segment of the global animal health market, accounting for 23% of market share. However, relatively few novel antiparasitic agents have been introduced into the market during recent decades. One exception, and a groundbreaking 21st century success story, are the isoxazolines, whose full potential has not yet been entirely explored. Unfortunately, resistance issues are present across most parasitic diseases, which generates a clear market need for novel resistance-breaking antiparasitics with new modes/mechanisms of action. Recent advances in science and technologies strongly suggest that the time is right to invest in new modalities such as parasitic vaccines or in environmentally friendly interventions.

Heartworm disease - Overview, intervention, and industry perspective.

Dirofilaria immitis, also known as heartworm, is a major parasitic threat for dogs and cats around the world. Because of its impact on the health and welfare of companion animals, heartworm disease is of huge veterinary and economic importance especially in North America, Europe, Asia and Australia. Within the animal health market many different heartworm preventive products are available, all of which contain active components of the same drug class, the macrocyclic lactones. In addition to compliance issues, such as under-dosing or irregular treatment intervals, the occurrence of drug-resistant heartworms within the populations in the Mississippi River areas adds to the failure of preventive treatments. The objective of this review is to provide an overview of the disease, summarize the current disease control measures and highlight potential new avenues and best practices for treatment and prevention.

The response of Mannheimia haemolytica to iron limitation: implications for the acquisition of iron in the bovine lung.

Mannheimia haemolytica is the major causative agent of shipping fever, a severe pneumonia in cattle causing high morbidity and mortality. A prerequisite of successful lung colonization by M. haemolytica is the necessity to adapt to the paucity of iron. The lack of genome information has precluded an assessment of the genetic repertoire available to M. haemolytica to adapt to low iron environments. To close this knowledge-gap, we have determined 90% of a virulent M. haemolytica serotype A1 genome sequence and produced a microarray in order to study gene expression under iron-limiting growth for 15, 30 and 60 min. M. haemolytica responded to iron limitation by the up-regulation of transcripts coding for receptors and ABC-type transporters of transferrin, haemoglobin, haem and siderophores. Real time PCR analysis of lung tissue from Mannheimia-infected calves demonstrated the in vivo transcription of two potential haemoglobin receptors, hmbR1 and hmbR2. The relative hmbR1 and hmbR2 transcript levels in the infected lung tissue were comparable to the induced levels observed under iron-limiting growth, demonstrating in vivo induction of receptor transcription in the context of an infection. When the iron response of M. haemolytica was compared to the iron response of Pasteurella multocida, another pathogen colonizing the bovine lung, only few homologous genes were induced in both organisms. These included the haemoglobin receptor hmbR2 and the periplasmic transport systems yfeABCD and fbpABC. The comparative analysis suggests that the two pathogens use different strategies to adapt to the iron-limiting environment in the bovine host.

How Phenotypic Screening Influenced Drug Discovery: Lessons from Five Years of Practice.

Since 2011, phenotypic screening has been a trend in the pharmaceutical industry as well as in academia. This renaissance was triggered by analyses that suggested that phenotypic screening is a superior strategy to discover first-in-class drugs. Despite these promises and considerable investments, pharmaceutical research organizations have encountered considerable challenges with the approach. Few success stories have emerged in the past 5 years and companies are questioning their investment in this area. In this contribution, we outline what we have learned about success factors and challenges of phenotypic screening. We then describe how our efforts in phenotypic screening have influenced our approach to drug discovery in general. We predict that concepts from phenotypic screening will be incorporated into target-based approaches and will thus remain influential beyond the current trend.

Blocking transmission of vector-borne diseases.

Vector-borne diseases are responsible for significant health problems in humans, as well as in companion and farm animals. Killing the vectors with ectoparasitic drugs before they have the opportunity to pass on their pathogens could be the ideal way to prevent vector borne diseases. Blocking of transmission might work when transmission is delayed during blood meal, as often happens in ticks. The recently described systemic isoxazolines have been shown to successfully prevent disease transmission under conditions of delayed pathogen transfer. However, if the pathogen is transmitted immediately at bite as it is the case with most insects, blocking transmission becomes only possible if ectoparasiticides prevent the vector from landing on or, at least, from biting the host. Chemical entities exhibiting repellent activity in addition to fast killing, like pyrethroids, could prevent pathogen transmission even in cases of immediate transfer. Successful blocking depends on effective action in the context of the extremely diverse life-cycles of vectors and vector-borne pathogens of medical and veterinary importance which are summarized in this review. This complexity leads to important parameters to consider for ectoparasiticide research and when considering the ideal drug profile for preventing disease transmission.

Complex molecules: do they add value?

The concept of complexity in chemistry has received much interest in the research community. Various measures to assess molecular complexity have been published, ranging from abstract complexity definitions to very specific application-oriented definitions. In this article we focus on molecular complexity in relation to biological activity. Connectivity and feature-based structural descriptors have been evaluated with reference to their potential as complexity measures. Our goal was to discuss the potential of the complexity concept to support the drug discovery process, helping to design suitable lead candidates. The studies have shown that highly active compounds, on average, are more complex than inactive compounds. However, complexity must be balanced with other molecular properties because more complex molecules have a higher probability to exhibit pharmacokinetic problems.

Quest for the rings. In silico exploration of ring universe to identify novel bioactive heteroaromatic scaffolds.

Bioactive molecules only contain a relatively limited number of unique ring types. To identify those ring properties and structural characteristics that are necessary for biological activity, a large virtual library of nearly 600 000 heteroaromatic scaffolds was created and characterized by calculated properties, including structural features, bioavailability descriptors, and quantum chemical parameters. A self-organizing neural network was used to cluster these scaffolds and to identify properties that best characterize bioactive ring systems. The analysis shows that bioactivity is very sparsely distributed within the scaffold property and structural space, forming only several relatively small, well-defined "bioactivity islands". Various possible applications of a large database of rings with calculated properties and bioactivity scores in the drug design and discovery process are discussed, including virtual screening, support for the design of combinatorial libraries, bioisosteric design, and scaffold hopping.

Identification and characterisation of the dopamine receptor II from the cat flea Ctenocephalides felis (CfDopRII).

G protein-coupled receptors (GPCRs) represent a protein family with a wide range of functions. Approximately 30% of human drug targets are GPCRs, illustrating their pharmaceutical relevance. In contrast, the knowledge about invertebrate GPCRs is limited and is mainly restricted to model organisms like Drosophila melanogaster and Caenorhabditis elegans. Especially in ectoparasites like ticks and fleas, only few GPCRs are characterised. From the cat flea Ctenocephalides felis, a relevant parasite of cats and dogs, no GPCRs are known so far. Thus, we performed a bioinformatic analysis of available insect GPCR sequences from the honeybee Apis mellifera, the mosquito Anopheles gambiae, the fruit fly Drosophila melanogaster and genomic sequences from insect species. Aim of this analysis was the identification of highly conserved GPCRs in order to clone orthologs of these candidates from Ctenocephalides felis. It was found that the dopamine receptor family revealed highest conservation levels and thus was chosen for further characterisation. In this work, the identification, full-length cloning and functional expression of the first GPCR from Ctenocephalides felis, the dopamine receptor II (CfDopRII), are described.

Isoxazolines: A Novel Chemotype Highly Effective on Ectoparasites.

Efficient control of arthropod ectoparasite infestations has a long-standing history in the agriculture and veterinary sectors, aiming to decrease the parasite burden of affected crops and animals. Ligand-gated chloride channels (LGCCs) modulated by γ-aminobutyric acid (GABA) and glutamate have been identified as suitable molecular targets, and several classes of potent parasiticides have been devised. Due to the increase in cross-resistance and decreased development of new chemical entities, an urgent need for new parasiticides or prevention schemes has emerged. In the last decade, an innovative isoxazoline chemotype appears to offer promise for inhibiting LGCCs with a new mode of action and distinct binding site from that of historical agents. Considerable efforts have focused on optimizing the antiparasitic activity of isoxazolines and may provide the potential for future human use.

Library design for fragment based screening.

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.151% observed in the identification of ligands with an IC(50) threshold in the micromolar range in an HTS setup and hitrates above or equal to 3% observed in NMR screening of fragments with an affinity threshold in the millimolar range. It is however necessary to keep in mind that the sets of target studied were not identical for both method and the experience in NMR screening is too limited for a final conclusion. The term hitrate as used here reflects only the success rate in the observation of ligand binding event. It must not be confused with the overall success rate of fragment and high throughput screening in the lead finding process, which can be entirely different, since the steps required to follow-up a ligand binding event to a lead are different for both methods. A survey of fragment-based lead discovery case studies given in the literature shows that in approximately half of the cases the initial hit fragment was discovered by screening a generic library, whereas in the other cases some knowledge about an initial ligands or the protein binding site has been used, whereas systematic virtual screening of fragment databases has been only rarely reported. As comparatively high hitrates were obtained, further consideration to optimize the generic fragment screening library were directed to the chemical tractability of the fragment. As several functional groups preferred by chemists for modification and linking of the fragments are also preferentially involved in interactions between the fragments and the target protein, a set of screening fragments was derived from chemical building blocks by masking its linker group by a chemical transformation which can be later on used in the chemical follow-up of the fragment hit. For example primary amines can be masked as acetamides. If the screening fragment is active the related building block can then be used for synthesis of a follow-up library.

Microarray analysis of Haemophilus parasuis gene expression under in vitro growth conditions mimicking the in vivo environment.

Haemophilus parasuis is the causative agent of polyserositis in pigs, a mostly fatal disease on the rise especially in early-weaned pigs and in pig herds with a high-health status. The mechanisms by which H. parasuis propagates through the body and colonizes the serous membranes are unknown. We have used an H. parasuis microarray to identify virulence genes involved in host adaptation. H. parasuis gene expression was analysed under in vitro growth conditions mimicking the environmental conditions encountered during an infection. These included iron-limitation, acidic and temperature stress and growth under microaerobic conditions. A kinetic impression of the gene regulation was obtained by analysing the transcription 10, 30 and 60 min after induction of the altered growth conditions. A total of 75 regulated H. parasuis genes were identified, most of which coded for transporters of iron and sugar metabolites, metabolic enzymes, DNA metabolism and hypothetical proteins with unknown functions. Furthermore, H. parasuis genes were identified that have homology to known virulence factors in other pathogenic bacteria. Homologues of some of the identified H. parasuis genes are known to be expressed during natural and experimental infections in pathogens of the Pasteurellaceae family.

Trypanothione reductase: a target protein for a combined in vitro and in silico screening approach.

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC50 values of <1 µM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC50 values down to 2 µM.

Linking phenotypes and modes of action through high-content screen fingerprints.

High-content screening (HCS) is a powerful technique for monitoring phenotypic responses to treatments on a cellular and subcellular level. Cellular phenotypes can be characterized by multivariate image readouts such as shape, intensity, or texture. The corresponding feature vectors can thus be defined as HCS fingerprints that serve as a powerful biological compound descriptor. Therefore, clustering or classification of HCS fingerprints across compound treatments allows for the identification of similarities in protein targets or pathways. We developed an HCS-based profiling panel that serves as basis for characterizing the mode of action of compounds. This panel measures phenotypic effects in six different compartments of U-2OS cells, namely the nucleus, the cytoplasm, the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. We profiled a set of 2,725 well-annotated compounds and clustered their corresponding HCS fingerprints to establish links between predominant cellular phenotypes and cellular processes and protein targets. We found various different clusters enriched for individual targets (e.g., HDAC, HSP90, TOP1, HMGCR, TUB), signaling pathways (e.g., PIK3/AKT/mTOR), or gene sets associated with diseases (e.g., psoriasis, leukemia). Based on this clustering we were able to identify novel compound-target associations for selected compounds such as a submicromolar inhibitory activity of Silmitasertib (a casein kinase inhibitor) on PI3K and mTOR.