A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

<div><p>Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an <i>in vivo</i> high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.</p></div

Functional classification and validation of selected anti-inflammatory hits.

<p>Anti-inflammatory candidates were classified in functional or therapeutic groups. Selected anti-inflammatory hit compounds exert dose-dependent anti-inflammatory effect on chemically induced inflammation. <b>(a)</b> Pie chart dividing the 70 anti-inflammatory hit compounds in functional or therapeutic groups containing at least 2 representatives (numbers). Groups with only one representative are summarized in “other”. (<b>b—e)</b> Dose-response behavior of selected anti-inflammatory hit compounds Tenatoprazole <b>(b)</b>, NECA <b>(c)</b>, Candesartan <b>(d)</b>, and 3-Bromo-7-nitroindazole <b>(e)</b>. Bar charts indicate means ± SEM of the initial inflammatory index (%) 90 minutes after CuSO₄ treatment (t = 0). Three individual experiments were performed with 15 replicate larvae for each condition, respectively. Statistics were evaluated with unpaired one-sided t-tests. ns–not significant: P > 0.05, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p

Experimental setup and compound categorization of the automated ChIn screen.

<p><b>(a)</b> Schematic diagram illustrating the screening strategy for compounds modulating inflammation. Individual larvae are distributed in 384-well plates. Drugs are added 1 h prior to treatment with CuSO₄ for 1h at 28°C in the dark followed by an exchange of medium and a 6h time-lapse analysis. Finally, screening data are automatically processed and analyzed. <b>(b)</b> Pie chart displaying the identified compound categories upon screening 1120 compounds of an FDA-approved library and the ICCB library of known bioactives. Numbers indicate percentages.</p

Inflammation induces S-nitrosylation in zebrafish larvae.

<p>Changes in total protein S-nitrosylation during inflammation was assessed with the modified biotin switch technique and detected by Western Blot. S-nitrosylated proteins in samples of control (lane 1) larvae and larvae with chemically induced inflammation (lane 2 and 3) were detected by anti-TMT immunoblotting. Chemically induced inflammation significantly induces S-nitrosylation, marked by additional strong anti-TMT immunoblotting signals (arrowheads), in protein extracts of CuSO₄ treated larvae. Inhibition of Nos with 250 μM L-NAME prior to CuSO₄ treatment prevents changes in protein S-nitrosylation. EF–1α was used as loading control.</p

Genetic knockdown of zebrafish <i>nos2b</i> and sGC (<i>gucy</i>) but not <i>nos1</i> and <i>nos2a</i> impair the inflammatory response.

<p>Assessment of the role of the 3 zebrafish Nos isoforms and sGC on peripheral sensory nervous system <b>(a)</b> and epithelial <b>(b)</b> inflammation upon genetic knockdown. <b>(a)</b><i>nos2b</i> and <i>gucy</i> knockdown significantly reduce initial inflammation at injured neuromasts as compared to wildtype (WT) larvae. <b>(b)</b> Knockdown of <i>nos2b</i> and <i>gucy</i> significantly reduce the number of neutrophils recruited to ventral fin wounds. Bar charts represent means of the initial inflammatory index (%) ± SEM 90 minutes after CuSO₄ treatment <b>(a)</b> and 1 h after mechanical injury of the ventral fin <b>(b)</b>. Three individual experiments were performed with 15 replicate morphant larvae for each condition, respectively. Statistics were evaluated with unpaired one-sided t-tests. ns–not significant: P > 0.05, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001.</p