Browsing by Subject "Drug Discovery"

Malaria is an infectious disease caused by apicomplexan Plasmodium species. These protozoan parasites are transmitted by a mosquito vector to a human host, wherein they undergo an asymptomatic liver stage followed by a symptomatic blood stage of infection. Despite eradication efforts, Plasmodium remain accountable for hundreds of thousands of mortalities per year, mostly caused by P. falciparum. The spreading resistance to the front-line antimalarials that broadly disrupt parasite proteostasis demands further characterization of their adaptive stress responses and novel multi-stage drug targets. This work focuses on the essential P. falciparum molecular chaperone heat shock protein 90 (PfHsp90). Specifically, PfHsp90 is expected to directly interact with a subset of parasite proteins to facilitate their ATP-dependent maturation, stabilization, and regulation. Despite this critical function, the scope of its chaperoning interactions—as well as its consequent contributions to mitigate cellular stress and maintain parasite proteome integrity throughout development—remains largely unresolved. To enable its functional interrogation, we first aimed to establish chemical inhibitors of PfHsp90 with greater affinity to the parasite compared to the conserved human homolog (HsHsp90). In general, our testing supports the particular utility of compounds that bind at the chaperone’s nucleotide-binding domain, as opposed to a putative C-terminal allosteric site, based on their high affinity and resolved mode of ATP-competitive inhibition. From this class of competitive inhibitors, we identified XL888 as exhibiting moderate selectivity to PfHsp90, despite that it was initially developed as a HsHsp90 inhibitor. Subsequent structural evaluation indicated that the PfHsp90 lid subdomain contributes to the parasite chaperone’s higher affinity interaction with XL888’s tropane scaffold. Considering this molecular basis, we were able to develop Tropane 1 as a novel XL888 analog with nanomolar affinity and approximately 10-fold selectivity to PfHsp90, which further demonstrated dual-stage, anti-Plasmodium activity. We next surveyed the PfHsp90-dependent proteome using innovative chemical biology strategies. Based on their thermal stability after chaperone inhibition, we identified 50 candidates as putative PfHsp90 interactors. A significant enrichment of proteasome regulatory particle components was represented in this analysis, from which we subsequently validated that PfHsp90 chaperones the 26S proteasome to support the controlled recycling of cellular proteins. Additionally, we adopted bio-orthogonal labeling with unnatural amino acids to track proteome dynamics in Plasmodium parasites. To date, we have implemented this approach to support that compromised PfHsp90 activity coordinates translation attenuation as a stress response. However, this work sets the foundation to employ such labeling to quantify PfHsp90-coordinated proteome dynamics across multiple parasite life stages. Collectively, these findings broaden our understanding of PfHsp90’s regulation of Plasmodium parasite proteostasis and further establish the potential of this molecular chaperone as a novel, multi-stage antimalarial drug target.