Identification of Potential Sepsis Therapeutic Drugs Using a Zebrafish Rapid Screening Approach

Abstract

In the military, combat wound infections can progress rapidly to life-threatening sepsis. The discovery of effective small-molecule drugs to prevent and/or treat sepsis is a priority. To identify potential sepsis drug candidates, we used an optimized larval zebrafish model of endotoxicity/sepsis to screen commercial libraries of drugs approved by the U.S. Food and Drug Administration (FDA) and other active pharmaceutical ingredients (APIs) known to affect pathways implicated in the initiation and progression of sepsis in humans (i.e., inflammation, mitochondrial dysfunction, coagulation, and apoptosis). We induced endotoxicity in 3- and 5-day post fertilization larval zebrafish (characterized by mortality and tail fin edema (vascular leakage)) by immersion exposure to 60 µg/mL Pseudomonas aeruginosa lipopolysaccharide (LPS) for 24 h, then screened for the rescue potential of 644 selected drugs at 10 µM through simultaneous exposure to LPS. After LPS exposure, we used a neurobehavioral assay (light-dark test) to further evaluate rescue from endotoxicity and to determine possible off-target drug side effects. We identified 29 drugs with > 60% rescue of tail edema and mortality. Three drugs (Ketanserin, Tegaserod, and Brexpiprazole) produced 100% rescue and did not differ from the controls in the light-dark test, suggesting a lack of off-target neurobehavioral effects. Further testing of these three drugs at a nearly 100% lethal concentration of Klebsiella pneumoniae LPS (45 µg/mL) showed 100% rescue from mortality and 88-100% mitigation against tail edema. The success of the three identified drugs in a zebrafish endotoxicity/sepsis model warrants further evaluation in mammalian sepsis models.

Keywords: drug discovery; endotoxicity; lipopolysaccharide; sepsis; zebrafish.

Figure 1

Caudal fin morphology of 3 dpf zebrafish. (A) Normal of caudal fin of control fish with no LPS exposure. (B) Caudal fin after 24 h of 60 µg/mL of P. aeruginosa LPS resulting in a tail edema and vascular leakage.

Figure 2

Dose response curves for 3 dpf zebrafish (top graph) and 5 dpf zebrafish (bottom graph) for 24-h exposures to LPS derived from E. coli 0111:B4, P. aeruginosa, and K. pneumoniae.

Figure 2

Dose response curves for 3 dpf zebrafish (top graph) and 5 dpf zebrafish (bottom graph) for 24-h exposures to LPS derived from E. coli 0111:B4, P. aeruginosa, and K. pneumoniae.

Figure 3

LPS concentration response curves for replicate tests during the optimization phase of assay development. The left graph depicts the percentage of fish with tail edema in response to increasing P. aeruginosa LPS concentrations. The right graph depicts the number of dead fish in response to increasing P. aeruginosa LPS concentrations. The goal was to determine a concentration that yielded close to 50% mortality while eliciting 100% combined tail edema and mortality in all fish. A concentration of 60 µg/mL of P. aeruginosa LPS was chosen for use in follow-on chemical screening.

Figure 4

Example of the rescue results from the initial drug screening from a single 96-well plate. Tegaserod showed complete rescue from LPS-induced tail and edema and mortality (n = 16 for each test condition). * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001.

Figure 5

Drug efficacy concentration range response curves for the top 16 rescue drugs. * = p ≤ 0.05, ** = p ≤ 0.01, and *** = p ≤ 0.001. n = 16 for each test condition.