Discovery of Novel Pain Therapies Without Side‐effects by Combining Targeted Mutagenesis and Phenotype‐based Drug Screening in Larval Zebrafish

Introduction Opioid drugs provide effective pain relief but come with considerable side‐effects including addiction and respiratory depression which can be lethal with overdose. During the COVID19 pandemic, the opioid epidemic reached almost 100,000 deaths in the United States, highlighting the urgent need to identify pain killers with reduced liability so they can be safely prescribed. One approach to develop safe pain killers is to combine existing opioid analgesics with drugs reducing respiratory depression. Using our novel biotechnology platforms in larval zebrafish, we propose to identify new molecular targets and drugs with potent analgesic properties, reduced respiratory liability, and without addictive properties. Methods Combining targeted mutagenesis and phenotype‐based drug screening approaches in larval zebrafish in vivo, we can quickly and effectively identify new molecular targets and screen new drug compounds to either target respiratory depression, analgesia ( Zaig and Montandon, 2021, eLife ), as well as addiction. We first screened customized drug libraries targeting voltage‐gated calcium channels and the adenylyl cyclase pathway, two molecular pathways involved in pain. We are also creating mutants using CRISPR‐cas9 in first generation zebrafish embryos to identify new molecular targets. Using our new models, we quantified respiratory depression by fentanyl in day post‐fertilization 7 larvae, as well as the nociceptive response to formalin. Results To determine the role of calcium channels opioid‐mediated effects, we combined fentanyl and the calcium channel activators nefiracetam (1µM) or FLP‐64176 (1µM). Both nefiractem and FLP‐64176 prevented respiratory depression by fentanyl (1µM). To target the adenylyl cyclase pathway, we combined fentanyl and the phosphodiesterase inhibitors pentoxifylline (50µM), roflumilast (1µM), or dipyridamole (100µM). Pentaxifylline prevented respiratory depression by fentanyl but not roflumilast and dipyridamole. To perform targeted mutagenesis, we injected 4 single guide RNAs targeting the orpm1 gene, i.e. the gene coding for µ‐opioid receptors. Six days later, we measured respiratory depression by fentanyl in knockout larvae or controls. In oprm1 ‐/‐ knockout larvae, respiratory depression by fentanyl was substantially reduced compared to controls, therefore confirming the validity of our targeted mutagenesis approach. We are currently targeting the genes coding for calcium channels and phosphodiesterases to determine whether they can be molecular targets for respiratory depression and/or analgesia. Discussion The successful identification of new drug combinations to prevent respiratory depression while preserving analgesia suggests that our drug discovery platform in larval zebrafish will substantially accelerate the identification of new opioid drugs with reduced morbidity and mortality. Our platform is also being leveraged to discover non‐opioid pain killers with reduced side‐effects.

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