Molecular targets of the anti-apicomplexan compound, tartrolon E

ABSTRACT: Development of effective and robust treatments for diseases caused by apicomplexan parasites is an ongoing medical and veterinary challenge. Since natural products are a proven source of effective and robust therapeutics against parasitic infections, we mined marine sources for anti-Toxoplasma and anti-Cryptosporidium activity. These screens identified a marine natural product, tartrolon E (trtE), with broad anti-apicomplexan activity in vitro and in vivo. Repeated attempts to select trtE-resistant T. gondii or P. falciparum mutants were unsuccessful implying that conservation of the trtE target is essential for parasite viability. To identify the target of trtE, we conducted a drug affinity response target stability (DARTS) assay. Proteins that were protected from proteolysis by the presence of trtE were identified by quantitative mass spectrometry. Two rhoptry proteins were highly enriched in trtE treated samples, TgROPLMF, and TgROP14. Both proteins are integral membrane proteins with a lipase maturation factor (LMF) domain. LMF-domain containing proteins are present in most all apicomplexans (ortholog group OG6_106580) consistent with trtE’s broad spectrum activity. The only apicomplexan lacking an obvious ortholog for the LMF domain containing proteins is Cryptosporidium. Our preliminary data lead us to hypothesize that TgROPLMF and TgROP14 are targets of trtE’s activity, and that trtE’s inhibition of the LMF domain proteins has downstream effects on proteins that interact with TgROPLMF. We will test these hypotheses through the completion of the following aims. Aim 1: To determine if the LMF domain proteins mediate the anti-parasitic activity of trtE: Toxoplasma parasites lacking TgROPLMF1, TgROP14, both proteins, or parasites overexpressing TgROPLMF, will be generated, validated and characterized. The susceptibility of the recombinant parasites to trtE, as compared to wild-type parasites, will be quantified in assays that evaluate inhibition of invasion and intracellular replication. Aim 2: To identify the interactome of TgROPLMF and TgROP14 and evaluate trtE’s ability to disrupt the TgROPLMF interactome. The interactome of the LMF domain containing proteins will be identified by proximity labeling with TurboID. Interacting proteins will be validated by endogenous tagging and tracking in wild type and recombinant parasites, and the ability of trtE to alter the TgROPLMF interactome tested. Aim 3: Identify putative trtE targets in Cryptosporidium parvum. Cryptosporidium is the only apicomplexan susceptible to trtE that lacks LMF domain proteins. In this aim our DARTS strategy will be repeated with sporozoites to identify putative targets in C. parvum. If the LMF domain proteins are trtE targets, we will have identified eminently druggable targets common to apicomplexan pathogens, providing lead compounds for new therapeutic strategies for diseases caused by apicomplexan parasites.