Combining B. thuringiensis crystal proteins with small molecule anthelmintics to combat parasitic nematode resistance - New treatments are urgently needed for parasitic gastrointestinal nematodes (GINs; hookworms, whipworms, roundworms) that infect ~1.5 billion people and cause significant morbidity in children, pregnant women, and working adults. The main drug used against GIN in mass drug administration (MDA) is albendazole (ABZ), with increased use of ivermectin (IVM) and consideration of the experimental drug emodepside (EMO). MDA with ABZ has not been curative, and IVM and EMO also have limitations. Thus, controlling GINs remains a critical unmet medical need. Experience with HIV, tuberculosis, and malaria shows that well-designed drug combinations can suppress resistance and enhance efficacy. Especially effective combinations are synergistic and show collateral sensitivity (CS), where resistance to drug A increases susceptibility to drug B. We have pioneered the development of crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) as safe, effective, and affordable next-generation anthelmintics to control GINs. For over 60 years, Cry proteins have controlled insects that vector disease and damage crops, including use in >100 million hectares of transgenic crops grown annually. Oral Cry5Ba treatment is safe for mammals and highly effective against GINs in rodents, dogs, sheep, pigs, and horses. We have now identified five other anthelmintic Cry proteins. Cry5Ba interacts synergistically and shows CS with nicotinic acetylcholine receptor agonist anthelmintics. Cry5Ba also synergizes with ABZ against all GINs and shows CS to EMO- and IVM-resistant nematodes. These data demonstrate that Cry proteins have excellent combinatorial properties with small molecule anthelmintic drugs. We propose to discover optimal combinations between six anthelmintic Cry proteins and three small molecule drugs (ABZ, IVM, or EMO) to suppress drug resistance and greatly improve efficacy. Three major aims will be pursued: Aim 1. Find strong synergistic interactions. Checkerboard design studies will be carried out with 6 Cry proteins x 3 small molecule drugs against three major GINs: Ancylostoma ceylanicum hookworms, Trichuris muris whipworms, and Ascaris suum roundworms. Synergy will be quantified with the ZIP synergy model. Aim 2. Find strong CS relationships. (1) Quantify the sensitivity of ABZ, IVM, and EMO-resistant Caenorhabditis elegans to six Cry proteins and the sensitivity of Cry-resistant C. elegans to ABZ, IVM, and EMO. (2) Quantify the sensitivity of ABZ- and IVM-resistant Haemonchus contortus to (a) Cry proteins and (b) Cry protein – small molecule drug combinations using a larval development assay. The data from Aims 1+2 will be integrated to design and test optimal Cry protein-drug combinations for in vivo synergy against parasitic infections, mechanistic studies using transcriptomics, and resistance management via computer modeling to optimize strategies for sustaining efficacy against GINs. The results will identify optimal combinations of Cry proteins and small molecule drugs to suppress drug resistance and increase efficacy, thereby providing durable relief from the human suffering caused by GINs.
