Repurposing and Designing Anti‐Giardial Drugs Targeting Lipids and Membranes
Authors: Pence Breanna , VanKoten Harrison , Klinkenberg James , Grajeda Brian , Ellis Cameron , Sirimulla Suman , Patterson Steven , Das Siddhartha 
Published: May 2022

Giardia lamblia is an intestinal parasite responsible for transmitting giardiasis, worldwide. This parasite exists in two morphologic forms: an infective cyst and a replicating trophozoite. The symptoms include diarrhea, vomiting, dehydration and stunted growth in children. Metronidazole (Mz) is a commonly prescribed medicine to treat giardiasis, however, the emergence of Mz resistant (Mzr) parasite is increasing rapidly. Therefore, it is imperative to identify new targets for developing non‐metronidazole classes of anti‐giardial agents. Our laboratory is interested in elucidating two novel targets for drug development against Giardia: (1) glucosylceramide transferase enzyme (gGlcT1), and (2) the lipid rafts/microvesicles assembly. Both targets are important for the attachment, growth, and encystation of Giardia and maintaining overall pathogenesis. For targeting gGlcT1, we took the ligand‐based approach to identify compounds against this sphingolipid synthesis enzyme. Our search identified several compounds (including FDA‐approved drugs) such as sugammadex, temocapril, delapril, and miglustat, which were active in blocking attachment of trophozoites and cyst formation in vitro. Another drug PPMP ((D‐threo‐1‐phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanol), a well‐known inhibitor of gGlcT1 with potential anti‐cancer activity, also inhibited trophozoite attachment (IC50,~6.5 mM) and Giardia cyst production (IC50,~3.5 mM). Previously, we have demonstrated that oseltamivir (Tamiflu®) was effective in disrupting giardial raft assembly and cyst production in vitro. In the current study, we synthesized eleven oseltamivir analogues and five of them were tested on the attachment of trophozoites. Oseltamivir and derivatives were synthesized from shikimic acid using standard methods. Briefly, 1x105 trophozoites were exposed to these analogues for 48 hours at concentrations of 0, 5, 10, and 20 µM on clear‐bottom 96‐well plates. Treated cells were washed with PBS (supplemented with 5 mM glucose and L‐cysteine) to remove non‐adhered cells. The attached parasites were stained with DAPI for 20 minutes at 37°C followed by quantification with the help of Biotek’s Cytation 5 Multimode Microplate Reader (GloMax). One analogue, called HVK‐ii‐43, caused 80% inhibition of attachment of trophozoites at 10 mM concentration. At the same concentration (10 mM), a second analogue, JK‐i‐45 also inhibited the attachment by 50% (IC50, ~ 10 mM). These results suggest that synthetic derivatives of oseltamivir are pharmacologically active against Giardiaand should be considered as effective anti‐giardial therapies. Our results also support the hypothesis that active compounds in combination doses or in hybrid forms (e.g., PPMP‐oseltamivir) could be used as novel anti‐giardial agents that will be effective against Mzr cells. Future work will involve gGlcT1 characterization, monitoring lipid rafts disassembly, and microvesicle release from Giardia after exposure to these and additional analogues.