30th Annual Meeting of the German Society for Parasitology

Prof.

Prof. Dr.

Targeting Schistosoma mansoni microRNA with small molecules to modulate host-parasite interactions (DDDS)

Abstract text Introduction Schistosoma is a genus of parasitic trematode responsible for causing schistosomiasis, this is perpetuated by an often chronic infection in which the parasite can persist for decades in the host. This persistence suggests a complex interaction where host factors are utilized for parasite development and the parasite modulates the host immune response[1]. One mechanism of immune modulation is through the use of microRNAs present in extracellular vesicles secreted by the parasites to regulate host immune cells. Schistosoma species mansoni, japonicum and hematobium, each produce a number of different microRNAs; with S. mansoni alone producing over 200 of these[2]. sma-miRNA-10 in particular has been shown to influence host T-cell fate determination through manipulation of the NF-κB pathway[3]. Objectives Our objective was to identify small molecules that can bind Schistosoma miRNA and interfere with this immunomodulation. Material &amp; methods We used a fragment-based screening approach by means of NMR to identify binders to the parasite microRNA-10. The small fragments identified were used to make larger elaborated molecules, which were then tested in cellular assays to confirm their ability to interfere with the action of sma-miR-10. Results We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated Schistosoma mansoni manipulation of the host immune system. These molecules were demonstrated in cellular assays to counteract NF-κB activity inhibition by S. mansoni miRNA-10. Conclusions This work suggests a potential role for such compounds in modulating host-pathogen interactions. More generally, these compounds offer a proof-of-concept as microRNA-targeting small molecules, that yield a distinct phenotype. [1] Y. Ofir-Birin, N. Regev-Rudzki, Science 2019, 363, 817-818. [2] L. Zhu, J. Liu, G. Cheng, Front Cell Infect Microbiol 2014, 4, 165-165. [3] T. Meningher, Y. Barsheshet, Y. Ofir‐Birin, D. Gold, B. Brant, E. Dekel, Y. Sidi, E. Schwartz, N. Regev‐Rudzki, O. Avni, D. Avni, EMBO reports 2020, 21.

Drug Development/Target Identification

Parasite-Host Interaction

Talk

Dr. rer. nat.

Treatment efficacy of oxfendazole and flubendazole against the rodent filaria Litomosoides sigmodontis is dependent on the immune system

Abstract text Introduction Filarial worms can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Control of both diseases occurs mainly via mass drug administration (MDA), but there are no drugs available that can eliminate the adult worms (&quot;macrofilaricide&quot;) using a short term treatment. Oxfendazole is a promising macrofilaricidal candidate with an improved oral availability in comparison to flubendazole and under preparation for phase 2 clinical trials in filariasis patients. Both benzimidazoles were tested in the Litomosoides sigmodontis mouse model, a model well established for preclinical drug development. Interestingly, several anti-filarial drugs require higher concentrations in vitro compared to in vivo to eliminate filariae. One possible reason for this discrepancy is that treatment efficacy of anti-filarial compounds is supported by the immune system. Objectives The aim of this study was to investigate the role of the immune system during treatment with two potential macrofilaricidal drugs, oxfendazole and flubendazole, and explore the potential to boost treatment efficacy via stimulation of the immune system. Materials &amp; Methods Wildtype (WT) BALB/c, eosinophil-deficient dblGATA, IL-4R/IL-5-/-, antibody-deficient µMT and B, T and NK cell-deficient RAG2/IL-2Rγ-/- mice were infected with L. sigmodontis. Mice harbouring adult worms (35 dpi) were treated with an optimal and suboptimal dose of flubendazole (FBZ) or oxfendazole (OXF) for up to 5 days. In a second part, WT mice were treated for two to three days with a combination of a suboptimal dose of OXF and IL-4, IL-5 or IL-33. Results At 70 dpi, WT mice displayed a reduction of the mean adult worm burden by &gt;94% compared to vehicle controls after both FBZ and OXF treatments. In contrast, treatment with either drug in knockout mice led to no reduction (RAG2/IL-2Rγ-/-) or a markedly lower reduction of the worm burden (dblGATA, IL-4R/IL-5-/-, µMT). The efficacy of a shortened treatment of OXF (-17.2% adult worms vs. vehicle) could be boosted to a 69.6% reduction via combination with IL-5, but not IL-4 or IL-33. Conclusions Our results suggest that various components of the immune system support the filaricidal effect of benzimidazoles in vivo and present an opportunity to boost treatment efficacy.

Effects of different knock-down resistance polymorphisms on pyrethroid susceptibility in the stable fly Stomoxys calcitrans and deep amplicon sequencing to screen for resistance haplotypes on German dairy farms

Abstract text Introduction Stable flies (Stomoxys calcitrans) are biting flies that disturb the well-being of livestock and severely affect their productivity. Excessive, decades-long use of pyrethroids against insects has resulted in widespread resistance, and we previously reported the presence of pyrethroid resistance in stable flies on farms in Brandenburg (BB). Pyrethroid resistance in stable flies has been associated with kdr (L1014F) and kdr-his (L1014H) mutations but not super-kdr (M918T) in the voltage-sensitive sodium channel (vssc) gene. Objectives This study aimed to examine the correlation between pyrethroid resistance and the vssc genotype (kdr and kdr-his) in stable flies and to screen German stable fly populations for kdr and kdr-his mutations. Materials &amp; methods In 2018, stable flies were collected from four dairy farms in BB and reared over two generations in the laboratory. The LD50 values for deltamethrin were determined for each isolate by topical application and compared with a susceptible laboratory strain (MSD). Selected, phenotypically tested flies were genotyped using PCR amplification of specific alleles (PASA). Using pooled stable fly samples collected from 65 dairy farms in BB, Schleswig-Holstein, and Baden-Württemberg in 2019, deep amplicon sequencing of the vssc gene was used to investigate the frequency and diversity of resistance alleles. Results The four tested field populations were identified as being phenotypically resistant to deltamethrin, with LD50 values of 18.0, 19.6, 40.5 and 47.0 ng deltamethrin/fly, 47.5 to 123.6-fold higher than for MSD strain. PASA showed the presence of kdr and kdr-his genotypes in all field populations, with significant (p &lt; 0.05) higher odds to survive treatment already in heterozygous individuals compared to wild-type flies, and stronger effects of kdr than kdr-his on the resistance phenotype. Deep amplicon sequencing revealed the presence of kdr and kdr-his variants on 100% of the farms with up to 55% of kdr and 85% of kdr-his alleles on individual farms. Phylogenetic analysis suggested at least two independent origins of both, kdr and kdr-his variants. In 19/65 pools, the world-wide first evidence for super-kdr alleles in stable flies (frequency 0.6-9.5%) was observed. Conclusion Pyrethroid resistance genotypes are widespread in stable fly populations on German dairy farms. The deep amplicon sequencing assays will allow to screen for pyrethroid resistance markers on a nation-level scale.

Drug Resistance

Vectors and Entomology

The value of P. falciparum field isolates for the prioritization of drug combinations to derisk malarial drug development: the case of Cabamiquine (M5717) – Pyronaridine

Abstract text Introduction Development and spreading of drug-resistant parasites substantially threaten malaria control and elimination. New antimalarial combination drugs are needed and required mandatory preclinical studies to determine the optimal combination partner, such as in vivo mice models and in vitro checkerboard assays. These models have been developed with long adapted P. falciparum laboratory strains leading to genetic modification and deletion of essential pathways of the parasites The translational value of these models to patient isolates is questionable and may explain the discrepancies observed between research and clinical trial data. This study is the first attempt to use freshly-derived P. falciparum isolates from patient to develop an in vitro checkerboard assay to assess drug-drug interaction (DDIs) using a promising new antimalarial combination of cabamiquine (M57171) and pyronaridine, soon entering Phase II. Methods Concentration and time-dependent in vitro cidial activity of cabamiquine and pyronaridine alone and in combination were determined against P. falciparum field isolates using standard 48 hours SYBR Green assay combined with Mitotracker readout. In vitro data were analyzed using non-linear mixed effects modelling describing the parasite growth and killing kinetics. Results For the first time in malaria, we used P. falciparum field isolates to assess DDIs using in vitro checkerboard assays. With cabamiquine and pyronaridine as experimental model, we successfully generate a range of EC50"s of the two drugs either alone or in combination. Moreover, we highlighted the importance of the combination in controlling possible adaptive resistance to one of the two drugs. The data were validated by using reference laboratory strains. Conclusions Being the first time that DDIs were studies using P. falciparum isolates directly from patient to inform the dose rate and dosing regiments of cabamiquine and pyrodnaridine in Phase II and III, offering a unique opportunity to validate the relevance of this new model. These data highlight the power of using non-adapted strains to better understand the future efficacy, resistance profile and safety of new antimalarial combination. If clinically confirmed these data are highlighting the central role that cutting edge African research needs to play in malaria control and eradication.

Prof

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation

Abstract text Introduction: The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to &quot;activation&quot; of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. Amino-artemisinins exhibit better activity against Toxoplasma gondii than artemisinin, however, the role of ROS has not been investigated. Materials and methods: T. gondii tachyzoites were grown in human foreskin fibroblasts. The T. gondii reference strain ME49 was treated with stepwise increasing amounts of artemisone and artemiside, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. The formation of reactive oxygen species (ROS) was assayed using dichlorofluorescein-diacetate (DCF-DA). Results: The amino-artemisinins are effective in vitro against the non-heme-degrading T. gondii tachyzoites with IC 50 values of 50–70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 μM) of these two compounds within few days. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and – to a lesser extent – in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. Conclusions: Amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium, possibly linked to TGME49_236950, and the rhoptry neck protein RON2.

The mRNA decapping enzyme of Trypanosoma brucei is a promising drug target

Abstract text Degradation of mRNAs is essential in all eukaryotes and contributes to the regulation of gene expression. The process usually starts with the removal of the poly(A) tail, is followed by the removal of the 5´ cap by a decapping complex and finishes by 5´-3´exoribonucleolytic decay. Kinetoplastids have conserved enzymes for the first and third reaction, but lack orthologues to all proteins of the decapping complex found in opisthokonts and plants. Instead, the Kinetoplastida decapping complex consists of the ApaH like phosphatase ALPH1 and several mostly Kinetoplastida-unique proteins: an example of convergent evolution. ALPH1 is a promising drug target: it is essential in Kinetoplastida, the entire enzyme family of ApaH like phosphatases does not exist in mammals and the enzyme can be purified in an active and soluble form. Here, we present the development of a high throughput enzyme assay that we will use to screen a variety of available drug libraries.

Workshop V
DDDS

Appointment

Chairs

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Workshop VDDDS

Appointment

Chairs

Programme

Workshop V
DDDS