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Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model

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Abstract

p21-activated kinase 1 (Pak1)—a key node protein kinase regulating various cellular process including angiogenesis—has been recognised to be a therapeutic target for multitude of diseases, and hence, various small molecule inhibitors targeting its activity have been tested. However, the direct toxic and anti-angiogenic effects of these pharmacologic agents have not been examined. In this study, we evaluate the translational efficacy of Pak1 inhibitor IPA-3 using zebrafish toxicity model system to stratify its anti-angiogenic potential and off-target effects to streamline the compound for further therapeutic usage. The morphometric analysis has shown explicit delay in hatching, tail bending, pericardial sac oedema and abnormal angiogenesis. We provide novel evidence that Pak1 inhibitor could act as anti-angiogenic agents by impeding the development of sub-intestinal vessel (SIV) and intersegmental vessels (ISVs) by suppressing the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), neurophilin 1 (NRP1) and its downstream genes matrix metalloproteinase (MMP)-2 and MMP-9. Knockdown studies using 2-O-methylated oligoribonucleotides targeting Pak1 also revealed similar phenotypes with inhibition of angiogenesis accompanied with deregulation of major angiogenic factor and cardiac-specific genes. Taken together, our findings indicate that Pak1 signalling facilitates enhanced angiogenesis and also advocated the design and use of small molecule inhibitors of Pak1 as potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting anomalous angiogenesis.

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Acknowledgments

We thank Dr. Sridhar Sivasubbu, IGIB for his kind support and advice. We also thank Samatha Mathew and Paras Sehgal, IGIB for their technical help to this work. The authors acknowledge the senior research fellowship (File No. 9/115(0774)/2014 EMR-I) by Council of Industrial and Scientific Research (CSIR), Government of India provided to SJ.

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Authors and Affiliations

  1. Department of Genetics, Dr. ALM PGIBMS, University of Madras, Chennai, 600 113, India

    Sankar Jagadeeshan, R. Vivek Sagayaraj, Navina Paneerselvan & Raghunathan Malathi

  2. Prism Foundation, Palissery P.O, Thrissur, 680 027, Kerala, India

    Syed Shabin Ghouse

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  1. Sankar Jagadeeshan
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  2. R. Vivek Sagayaraj
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  3. Navina Paneerselvan
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  4. Syed Shabin Ghouse
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  5. Raghunathan Malathi
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Correspondence to Raghunathan Malathi.

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Figure S1

Representative image showing pericardial oedema and linear heart tube with increasing concentration of IPA-3. (PPTX 6349 kb)

Figure S2: A- Real time PCR analysis of Pak1, Pak2 and Vegf gene in control-2OMe, Pak1-2OMe and Pak2-2OMe (0.25 mM) injected embryos. B- Representative images of Pak2-OMe injected embryos with cranial haemorrhage. Graph showing percentage of survived embryos with cranial haemorrhage. Graph showing C-survival rate, percentage of embryos D- with pericardial oedema and E- with defective heart.

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Jagadeeshan, S., Sagayaraj, R.V., Paneerselvan, N. et al. Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model. Cell Biol Toxicol 33, 41–56 (2017). https://doi.org/10.1007/s10565-016-9358-5

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  • DOI: https://doi.org/10.1007/s10565-016-9358-5

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