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PCL474: Pilot Zebrafish Chemical Genetic Screen for Novel Anti-Inflammatory Drugs
Xi (Tina) Yuan
999066718
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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
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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
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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
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Therefore, if we can prevent the migration of neutrophils, then we are able to avoid
persistent inflammation.
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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.
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Experiment Design
Induction of inflammatio
n Read-out Pilot Drug
Screen
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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.
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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.
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Problems to be addressed
1. DCF-DA- optimizing dosage2. DCF-DA- optimizing time points3. Validations with positive controls
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DCF-DA- optimizing dosage
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DCF-DA- optimizing dosage
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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
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Positive Control: Diclofenac inhibit ROS generation
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Positive Control: Ibuprofen inhibit ROS generation
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Diclofenac and Ibuprofen reduce neutrophil migration to wound site
Control
Ibuprofen
Diclofenac
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0hpi: tailfin transection + addition of crude microbial extracts (compound library)
5hpi: addition of DCFH-DA dye (100uM)
6hpi: Fluoresence Microscopy
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Pilot screen of 20 compounds: A1 & C3 as potential hits
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Validation experiment: A1 confirmed to be effective
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Future Goal 1. Dose response for A1 2. Validate hits with neutrophil numbers 3. Screen more compounds
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Questions and Comments
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Acknowledgements
• Dr. Xiao-Yan Wen• Anju Philips • Youdong Wang • Peter Zhou • Zhouduo Wang • Xiaohua Liu• Koroboshka Brand