Zebrafish Chemical Screen for novel anti-inflammatory drugs - a phenotype-based approach

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