Biology subjects

Hanson, K. K.

Publications and source records attributed to Hanson, K. K..

3 recordsLinked to original sources

bioRxiv2024

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be multistage actives, capable of killing parasites in the liver and blood, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits, both of which are parasite-specific quinoline-4-carboxamides, and analogues of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

microbiology↗

bioRxiv2023

Translation inhibition efficacy does not determine the Plasmodium berghei liver stage antiplasmodial efficacy of protein synthesis inhibitors

Protein synthesis is a core cellular process, necessary throughout the complex lifecycle of Plasmodium parasites, thus specific translation inhibitors would be a valuable class of antimalarial drugs, capable of both treating symptomatic infections in the blood and providing chemoprotection by targeting the initial parasite population in the liver, preventing both human disease and parasite transmission back to the mosquito host. As increasing numbers of antiplasmodial compounds are identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of molecular target or mechanism, it would be useful to gain deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential. Here, we probed that relationship using the P. berghei-HepG2 liver stage infection model. Using o-propargyl puromycin-based labeling of the nascent proteome in P. berghei-infected HepG2 monolayers coupled with automated confocal feedback microscopy to generate unbiased, single parasite image sets of P. berghei liver stage translation, we determined translation inhibition EC50s for five compounds, encompassing parasite-specific aminoacyl tRNA synthetase inhibitors, compounds targeting the ribosome in both host and parasite, as well as DDD107498, which targets Plasmodium eEF2, and is a leading antimalarial candidate compound being clinically developed as cabamiquine. Compounds were then tested at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, with parasites followed up to 120 hours post-infection to assess liver stage antiplasmodial effects of the treatment. Our data conclusively show that translation inhibition efficacy per se does not determine a translation inhibitors antiplasmodial efficacy. DDD107498 was the least effective translation inhibitor, yet exerted the strongest antimalarial effects at both 5x- and 10x EC50 concentrations. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to release of hepatic merozoites for all compound, demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites, and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy cannot function as a proxy for antiplasmodial effectiveness, and highlight the importance of exploring the ultimate, as well as proximate, mechanisms of action of these compounds on liver stage parasites.

microbiology↗

bioRxiv2023

Single-cell quantitative bioimaging of P. berghei liver stage translation.

Plasmodium parasite resistance to existing antimalarial drugs poses a devastating threat to the lives of many who depend on their efficacy. New antimalarial drugs and novel drug targets are in critical need, along with novel assays to accelerate their identification. Given the essentiality of protein synthesis throughout the complex parasite lifecycle, translation inhibitors are a promising drug class, capable of targeting the disease-causing blood stage of infection, as well as the asymptomatic liver stage, a crucial target for prophylaxis. To identify compounds capable of inhibiting liver stage parasite translation, we developed an assay to visualize and quantify translation in the P. berghei-HepG2 infection model. After labeling infected monolayers with o- propargyl puromycin (OPP), a functionalized analog of puromycin permitting subsequent bioorthogonal addition of a fluorophore to each OPP-terminated nascent polypetide, we use automated confocal feedback microscopy followed by batch image segmentation and feature extraction to visualize and quantify the nascent proteome in individual P. berghei liver stage parasites and host cells simultaneously. After validation, we demonstrate specific, concentration-dependent liver stage translation inhibition by both parasite-selective and pan-eukaryotic active compounds, and further show that acute pre-treatment and competition modes of the OPP assay can distinguish between direct and indirect translation inhibitors. We identify a Malaria Box compound, MMV019266, as a direct translation inhibitor in P. berghei liver stages and confirm this potential mode of action in P. falciparum asexual blood stages.

microbiology↗