Peg 400

Compounds, originally from the National Cancer Institute Diversity Set VI chemical library, were procured from different chemical vendors: NSC234945 and NSC10447 were obtained from A2B Chem LLC, USA; NSC158011 and NSC252172 were acquired from AKos Consulting & Solutions GmbH, Germany; and NSC303244 and NSC638080 were purchased from ChemSpace LLC, USA. All compounds were ≥95% pure as determined by liquid chromatography-mass spectrometry and/or proton nuclear magnetic resonance analysis and were shipped as powder. Upon receipt, compounds were individually reconstituted in cell culture grade DMSO (ATCC) and stored as stock solutions at −20°C. For in vitro experiments, stock solutions of compounds and their combinations were diluted in RPMI-1640 medium to produce working solutions for testing cytotoxicity and anti-cryptosporidial efficacy. For in vivo studies, stock solutions of individual compounds and their combinations were diluted in 100% (vol/vol) polyethylene glycol 400 (PEG 400; Rigaku Reagents) to a final DMSO concentration of 25%. Paromomycin sulfate (Thermo Scientific) was used as a control for in vitro and in vivo studies because of its previously reported activity against C. parvum (22 (link), 40 (link)).

l-Canavanine, tris(2-carboxyethyl)phosphine
(TCEP), l-Arg, sodium phosphate monobasic, and imidazole
were acquired from Sigma (St. Louis, Missouri). Kanamycin, isopropyl
β-d-1-thiogalactopyranoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), and Ni-NTA agarose beads were purchased from GoldBio
(St. Louis, Missouri). 2OG was purchased from Fluka. EDTA was purchased
from Invitrogen. 50% PEG 3350, PEG 400, PEG MME 550, and 50% ethylene
glycol were purchased from Rigaku or Hampton. All other chemicals
were reagent grade or better.