A rapid sporozoite viability assay identifies anti- <i>Cryptosporidium parvum</i> leads and targetable enzymatic activities in the invasive stage

<jats:title>Abstract</jats:title> <jats:p> Current phenotypic screens for anti- <jats:italic>Cryptosporidium</jats:italic> compounds typically quantify intracellular parasite growth in host cell cultures after two days of infection. Here, we developed a rapid, host cell-free phenotypic assay that directly measures compound-induced loss of viability in excysted <jats:italic>Cryptosporidium parvum</jats:italic> sporozoites, the invasive stage that initiates infection. We first compared qRT-PCR, luminescence ATP, and resazurin fluorescence readouts for detecting viable sporozoites. The luminescence ATP assay provided the best balance of linear dynamic range, assay time, parasite input, and cost, and was therefore adapted for high-throughput screening. </jats:p> <jats:p> Screening 5,000 bioactive compounds at 40 μM identified 28 primary hits with ≥50% inhibition of sporozoite viability, including 14 with &gt;60% inhibition. Secondary screening of these 14 compounds at 4 μM identified five hits retaining &gt;50% inhibition: ZL0420, sulbactam, abexinostat, kojic acid, and SIB-1757. All five showed submicromolar activity against free sporozoites, with EC <jats:sub>50</jats:sub> values of 0.073–0.311 μM. Four compounds, abexinostat, ZL0420, SIB-1757, and sulbactam, also inhibited intracellular parasite growth in vitro, with EC <jats:sub>50</jats:sub> values of 0.316–11.87 μM and selectivity indices from &gt;17 to &gt;107. In an IFN-γ-knockout mouse model, these four compounds reduced oocyst shedding over the 35-day experiment by 53.7–79.0% based on area-under-the-curve analysis and improved body-weight trajectories and ileal histopathology. </jats:p> <jats:p> Biochemical assays further showed that abexinostat inhibited native parasite HDAC activity at low nanomolar concentrations, while sulbactam inhibited a β-lactamase-like activity in sporozoite lysates. These findings establish sporozoite viability as a rapid screening endpoint and identify anti- <jats:italic>Cryptosporidium</jats:italic> leads associated with targetable enzymatic activities in the invasive stage. </jats:p> <jats:sec> <jats:title>Author summary</jats:title> <jats:p> <jats:italic>Cryptosporidium parvum</jats:italic> is a major cause of diarrheal disease in humans and young animals, but treatment options remain limited. Most laboratory screens for new drugs against this parasite require infection of host cells and measurement of parasite growth after one or more days. We developed a faster approach that tests whether compounds can directly damage freshly excysted sporozoites — the parasite stage that first invades intestinal cells — or reduce their viability. This assay can be completed within a few hours and does not require host cells. </jats:p> <jats:p> Using this approach, we screened 5,000 bioactive compounds and identified several molecules that rapidly reduced sporozoite viability. Four of these compounds also inhibited parasite growth in cell culture and reduced infection severity in a mouse model, as measured by parasite shedding, body-weight changes, and intestinal pathology. We further showed that one compound inhibits parasite histone deacetylase activity, while another inhibits a β-lactamase-like activity present in sporozoites. Our study provides a rapid screening strategy and highlights vulnerable biological activities in the invasive stage of <jats:italic>C. parvum</jats:italic> . </jats:p> </jats:sec>