The assessment of a protease -dependent prodrug in the nematode Caenorhabditis elegans for the treatment of parasitic helminth infections

Oluwadare, Eyitayo; Turnbull, Agnes; Malone, Eva; Mincher, David; Proudfoot, Lorna

Abstract

Billions of livestock, and a third of the current human population, are infected with helminth parasites. In livestock, these helminth infections are accompanied by significant morbidity and mortality, and although anthelmintic drugs are available, the development of resistance to the drugs poses a threat to their continuous use. Therefore, there is a need to develop more effective and better targeted anthelmintics that would combat resistance.
The nematode helminth (worm) Caenorhabditis elegans was used as a representative model of helminth parasites which utilize proteases present in parasites, and these proteases could be novel targets for anthelmintic drugs. Proteases hydrolyse peptide bonds which link amino acids, and this characteristic is exploited using a prodrug approach to deliver an active anthelmintic compound to the helminth.
We employed the use of a novel synthetic molecular probe to detect the presence of a protease in worms. The probe consists of a rhodamine fluorophore and an asparagine-containing peptide attached to a quencher. Cleavage by the protease separates the quencher from the probe triggering fluorescence. Relative fluorescence intensity was used as a measure of protease activity detected in C. elegans homogenates exposed to 10µM probe after 2 hours incubation (20oC) at pH5. The excitation and emission wavelength for the analysis were 544nm and 590nm respectively. Subsequently, using differential solubilization techniques, we suggest that the helminth protease is membrane-bound following observation of its activity in somatic fractions exposed to the probe under the same conditions as described above.
Furthermore, results show that live worms incubated with rhodamine fluoresce after 18 hours exposure at concentrations as low as 100nM with no significant lethality on the worms. Rhodamine may act as a carrier to aid delivery of entire probe prodrugs into live worms. The success of this rhodamine-aided prodrug delivery would take us a step further in being able to exploit proteolytic activity in the worm to release the anthelmintic drug following the protease cleavage. This would aid in the development of new anthelmintics and improve susceptibility of parasites to currently used ones by potentially evading anthelmintic resistance mechanisms such as efflux-mediated resistance.

Citation

Oluwadare, E., Turnbull, A., Malone, E., Mincher, D., & Proudfoot, L. (2015, August). The assessment of a protease -dependent prodrug in the nematode Caenorhabditis elegans for the treatment of parasitic helminth infections. Poster presented at Molecular & Cellular Biology of Helminth Parasites IX, Hydra, Greece

• Presentation Conference Type: Poster
• Conference Name: Molecular & Cellular Biology of Helminth Parasites IX
• Start Date: Aug 31, 2015
• End Date: Sep 5, 2015
• Deposit Date: Jul 19, 2023