A comparative analysis of Hsp90 and its possible role in drug resistance in nematode parasites

Lead Research Organisation: University of Glasgow
Department Name: Veterinary Infection and Immunity

Abstract

Nematode parasites are round worms that infect humans and domestic animals. They can cause illness and a variety of symptoms in their hosts and are very important both economically in farm animals and in public health terms, particularly in poorer areas of the world. A major problem with nematode parasites of farm animals is the spread of drug resistance. There are three different types of drugs available to treat infected animals, but some nematodes are now resistant to all three classes of drug. In this study we will investigate whether a protein called Hsp90 is a good target for new drugs. This protein is of great interest in the field of cancer research and many new compounds are being prepared which inhibit its function. In addition we will test a hypothesis that Hsp90 might also be involved in drug resistance in nematodes. It has recently been shown to be very important in fungi that have become resistant to drugs and we will investigate whether it may have a similar function in nematodes. As there are many new inhibitors of Hsp90, this approach could offer the potential to reverse drug resistance.

Technical Summary

Hsp90 is unique amongst the family of heat shock proteins because of the nature of the molecules with which it interacts. These include a variety of kinases and receptors which are essential for survival. Geldanamycin (GA), a compound which inhibits Hsp90 activity, has been widely used to define Hsp90 interactions and function. The free-living model nematode C. elegans is unique amongst eukaryotes in expressing a GA-resistant Hsp90. The first aim of this application is to investigate whether C. elegans is the exception or the norm amongst nematodes by analysing Hsp90 in a variety of important parasitic nematodes. Nematode parasites will be classified as GA-resistant or susceptible and amino acids associated with resistance identified and targetted for site directed mutagenesis. A transgenic line of C. elegans expressing mutated Hsp90 will be prepared and assayed for GA-susceptibility. The second aim of the grant is to investigate whether Hsp90 plays a role in drug resistance in nematodes. Here we propose to use C. elegans mutants that are resistant to anthelmintic compounds and determine whether a resistant phenotype can be modulated by compromising Hsp90 function. As there are an increasing number of small molecule inhibitors of Hsp90 becoming available, this study has the potential to offer a novel approach to restricting the spread of drug resistance.

Publications

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Devaney E (2010) microRNAs: a role in drug resistance in parasitic nematodes? in Trends in parasitology

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Devaney E (2011) Molecular and physiological basis of nematode survival

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Devaney E (2016) Hsp90 Inhibitors in Parasitic Nematodes: Prospects and Challenges. in Current topics in medicinal chemistry

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Gillan V (2014) A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes in PLoS Neglected Tropical Diseases

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Gillan V (2014) Nematode Hsp90: highly conserved but functionally diverse. in Parasitology

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Him NA (2009) Hsp-90 and the biology of nematodes. in BMC evolutionary biology

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Taldone T (2010) Assay strategies for the discovery and validation of therapeutics targeting Brugia pahangi Hsp90. in PLoS neglected tropical diseases

 
Description Nematode parasites are roundworms that infect humans and domestic animals. They cause illness in their hosts and are important in both farm animals and humans. A major problem with nematode parasites of farm animals is the spread of drug resistance. Three different types of drugs are available to treat infected animals, but some nematodes are resistant to all three classes. We aimed to investigate the function of a protein called heat shock protein 90 (Hsp90) in nematodes, and specifically in drug resistance. Hsp90 is important in fungi that have become resistant to drugs, with inhibition of Hsp90 reversing resistance. The first part of the project investigated why some nematodes were susceptible to geldanamycin (GA), an Hsp90 inhibitor, while others are not. We surveyed Hsp90 from 24 different free-living or parasitic nematodes for GA-binding and discovered that GA-binding is associated with life history: free-living worms or parasites that have a free-living stage in the environment do not bind GA. In contrast Hsp90 from obligate parasites and those worms in which the free-living stage is enclosed within a resistant eggshell bind GA. These findings support the hypothesis that nematode hsp90 genes are undergoing adaptive evolution: hsp90 is an essential gene, therefore exposure to inhibitors is harmful. GA is synthesised by soil-dwelling microorganisms, thus worms that live in the environment could be exposed to GA and may have evolved GA-resistant hsp90. We investigated whether Hsp90 played a role in drug resistance in nematodes using the model nematode C. elegans. We assessed Hsp90 levels in C. elegans mutants showing resistance to ivermectin (IVM), benzimidazoles compounds or levamisole, but no significant differences were observed. To study drug resistant worms that are more similar to field isolates of parasitic nematodes, we used additional lines of IVM-resistant C. elegans, isolated by continuous growth on drug. We used RNA interference (RNAi) to knock down hsp90 levels to determine whether a reduction in Hsp90 affects sensitivity to IVM. Hsp90 levels were consistently reduced by 40-50%. Control or RNAi-treated worms were exposed to IVM and viability assessed. The results were variable: in some experiments, a difference was observed in survival in IVM between resistant worms exposed to RNAi and control worms, indicating that hsp90 may play a role in the ability of C. elegans to tolerate drug. However despite repeating these experiments on numerous occasions the results were not reproducible. The successful knock-down of hsp90 in C. elegans using RNAi prompted us to attempt similar experiments with H. contortus. We selected three separate regions of the H. contortus hsp90 gene as a target for RNAi knockdown, but were unable to achieve knockdown of the hsp90 mRNA. Thus we could not prove that Hsp90 was involved in drug resistance. In addition to Hsp90, miRNAs are involved in drug resistance in some systems, including some tumor cells. We investigated the possible role of miRNAs in IVM resistant C. elegans using microarrays and showed that a small number of microRNAs were differentially expressed. These are now the focus of further study.
Exploitation Route The role of Hsp90 in drug resistance remains unresolved but is a focus of interest in other labs. The work carried out under this project stimulated an interest in repurposing of Hsp90 inhibitors for neglected tropical diseases and helped develop an interest in anthelmintic resistance in my lab which has led to further studies and grant income. In addition our work led to a new collaboration with cancer biologists at the Memorial Sloan Kettering Cancer Center, NYC.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
URL http://www.nature.com/nm/journal/v17/n6/full/nm0611-646.html
 
Description Mostly by other researchers in the field as our work and the publications stemming from it generated considerable interest in the complexities of Hsp90 and the mechanisms by which particular classes of inhibitors bind. Our work resulted in a new collaboration with a leading cancer biology group based at Sloan Kettering Memorial Cancer Center in New York.
First Year Of Impact 2010
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic