Leonie Konopka’s research while affiliated with Philipps University of Marburg and other places

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditiselegans and Leishmaniaamazonensis and predicted sensitivity in Aedes sp., Schistosomamansoni, Trypanosomabrucei, Plasmodiumfalciparum, and Toxoplasmagondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

Selective inhibition of eukaryotic initiation factor 4A (eIF4A), an RNA helicase, has been proposed as a strategy to fight pathogens. Plant-derived rocaglates exhibit some of the highest specificities among eIF4A inhibitors. Sensitivity to rocaglates is determined by key amino acid (aa) residues mediating reversible clamping of the eIF4A:RNA complex. To date, no comprehensive assessment of eIF4A sensitivity to rocaglates across the eukaryotic tree of life has been performed to determine their anti-pathogenic potential. We performed an in silico analysis of the substitution patterns of six aa residues in eIF4A1 critical to rocaglate binding (human positions 158, 159, 163, 192, 195, 199), uncovering 35 pattern variants among 365 eIF4As sequenced to date. In silico molecular docking analysis of the eIF4A:RNA:rocaglate complexes of the 35 variants, modeled in a human eIF4A environment, and in vitro thermal shift assays with recombinantly expressed human eIF4A mutants, representing select natural and artificial variants, revealed that sensitivity to a natural or one of two synthetic rocaglates—silvestrol, CR-1-31-B, or zotatifin—was associated with lower inferred binding energies and higher melting temperature shifts. Helicase activities were comparable across variants and independent of sensitivity to rocaglates. In vitro testing with silvestrol validated predicted resistance based on position 163 substitutions in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp. , Schistosoma mansoni , Trypanosoma brucei , Plasmodium falciparum , and Toxoplasma gondii . Our analysis shows resistance to rocaglates emerging in disparate eukaryotic clades pointing to resistance being a selective neutral trait except in rocaglate-producing Aglaia plants and their fungal parasite Ophiocordyceps . The analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens including Galleria mellonella , Ustilago maydis , Babesia ovata , and Cryptosporidium sp. , with rocaglates. Finally, combined docking and thermal shift analyses might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens. Author Summary In the ongoing search for novel ways to fight non-viral and non-bacterial pathogens, targeting translation—the universal process of protein synthesis—to inhibit growth and cell proliferation has emerged as an attractive strategy. Here, we focused on the potential of rocaglates, a group of plant-derived compounds, to inhibit an early step in translation mediated by a RNA helicase called eIF4A. We performed a comprehensive analysis of eIF4A sequence variants to determine their potential sensitivities to rocaglates, especially in pathogens of prokaryotic, fungal, or animal origin. We complemented this in silico analysis with enzyme-based in vitro and whole pathogen in vivo experiments to confirm the sensitivity or resistance to rocaglates of specific variants of eIF4A. Our analysis provides the first comprehensive picture of rocaglate sensitivity among pathogens and establishes targeting important insect, plant, animal, and human pathogens such as wax moth larvae, a major parasite of honey bees, corn smut, a widely distributed fungal disease, Babesia , a livestock parasite that causes anemia and babesiois, and Cryptosporidium , the causative organism of cryptosporidiosis in humans, with rocaglates as a viable anti-pathogen strategy.

Schistosomiasis is a neglected tropical disease caused by blood flukes of the genus Schistosoma and causes severe morbidity in infected patients. In 2018, 290.8 million people required treatment, and 200,000 deaths are reported per year. Treatment of this disease depends on a single drug, praziquantel (PZQ). However, in the past few years, reduced sensitivity of the parasites toward PZQ has been reported. Therefore, there is an urgent need for new drugs against this disease. In the past few years, we have focused on a new substance class called biaryl alkyl carboxylic acid derivatives, which showed promising antischistosomal activity in vitro. Structure–activity relationship (SAR) studies of the carboxylic acid moiety led to three promising carboxylic amides (morpholine, thiomorpholine, and methyl sulfonyl piperazine) with an antischistosomal activity down to 10 µM (morpholine derivative) and no cytotoxicity up to 100 µM. Here, we show our continued work on this substance class. We investigated, in extended SAR studies, whether modification of the linker and the thiophene ring could improve the antischistosomal activity. We found that the exchange of the alkyl linker by a pentadienyl or benzyl linker was tolerated and led to similar antischistosomal effects, whereas the exchange of the thiophene ring was not tolerated. Our data suggest that the thiophene ring is important for the antischistosomal activity of this compound class. Biaryl alkyl carboxylic acid derivatives show antischistosomal activity in vitro. Extended structure–activity relationship studies were used to investigate the effects of modifications of the linker and the thiophene ring on the activity. It was found that replacement of the alkyl linker by more rigid structures is tolerated whereas replacement of the thiophene in the biaryl moiety leads to complete loss of the antischistosomal activity.