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PCL474: Pilot Zebrafish Chemical Genetic Screen for Novel Anti-Inflammatory Drugs
Xi (Tina) Yuan
999066718
Overview1. Introduction 2. Methods and Materials3. Results and Discussion
1. Established Screening Assay 1. MPX Mcherry & DCFH-DA2. MPX GFP & neutrophil counts
2. Chemical Screening of 20 compounds 3. Validation of hit compounds
4. Future goal
Neutrophil and the innate immune system
• Neutrophils are the first line of innate immune defense against infection or injuries. – migrate toward the site of the inflammation
through chemotaxis. • Through the process of destroying pathogens,
our immune system sometimes induces changes in tissue, characterized by redness, warmth, swelling and pain. These tissue changes are called inflammation
Neutrophil and the innate immune system
• Normally, resolution of inflammation occurs as soon as the threat of infection abates.
• However, incomplete resolution of the inflammatory process damages the surrounding tissue and contributes to chronic diseases – Atherosclerosis – Chronic obstructive pulmonary disease – Sepsis
• Many such diseases, respond poorly to conventional treatments
Therefore, if we can prevent the migration of neutrophils, then we are able to avoid
persistent inflammation.
Zebrafish are ideal model for phenotypic drug screening
• Conservation of the principle components of the innate immune system
• Small size, • High fecundity rates • Rapid development • Optical transparency• Cost-effective
Tat J, Liu M, Wen X-Y. Zebrafish cancer and metastasis models for in vivo drug discovery. Drug Discov Today Technol. 2013;10(1):e83-e89. doi:10.1016/j.ddtec.2012.04.006.
Experiment Design
Induction of inflammatio
n Read-out Pilot Drug
Screen
Readout #1: neutrophil migration-Fin transection
• transection of the caudal fin in 3 days post-fertilization (dpf) mpx:GFP/mpx:mcherry larvae
• Injured larvae are exposed to compounds from 4 hours post-injury (hpi) to 12 hpi. – Positive control: known inhibitors of inflammatory pathways – Negative control: DMSO
Robertson AL, et al. A zebrafish compound screen reveals modulation of neutrophil reverse migration as an anti-inflammatory mechanism. Sci. Transl. Med. 2014;6(225):225ra29. doi:10.1126/scitranslmed.3007672.
Readout #2: ROS Reporter Assay
• 29, 79-dichlorodihydrofluorescein-diacetate (DCFH-DA) can be oxidized by ROS to form DCF (green fluorescence)
Nauseef, William M., and Niels Borregaard. "Neutrophils at work." Nature immunology 15.7 (2014): 602-611.
Problems to be addressed
1. DCF-DA- optimizing dosage2. DCF-DA- optimizing time points3. Validations with positive controls
DCF-DA- optimizing dosage
DCF-DA- optimizing dosage
Positive Control: Ibuprofen and diclofenac
• Belongs to the class of nonsteroidal anti-inflammatory drugs (NSAID)
• They exerts anti-inflammatory effects via inhibition of prostaglandin synthesis
Positive Control: Diclofenac inhibit ROS generation
Positive Control: Ibuprofen inhibit ROS generation
Diclofenac and Ibuprofen reduce neutrophil migration to wound site
Control
Ibuprofen
Diclofenac
0hpi: tailfin transection + addition of crude microbial extracts (compound library)
5hpi: addition of DCFH-DA dye (100uM)
6hpi: Fluoresence Microscopy
Pilot screen of 20 compounds: A1 & C3 as potential hits
Validation experiment: A1 confirmed to be effective
Future Goal 1. Dose response for A1 2. Validate hits with neutrophil numbers 3. Screen more compounds
Questions and Comments
Acknowledgements
• Dr. Xiao-Yan Wen• Anju Philips • Youdong Wang • Peter Zhou • Zhouduo Wang • Xiaohua Liu• Koroboshka Brand