Background

Tetrahydrobiopterin (BH4) is a co-factor metabolite used by the aromatic amino acid hydroxylases and nitric oxide synthases in the production of dopamine, serotonin, noradrenaline, and nitric oxide (Werner et al., 2011). BH4 synthesis is increased in dorsal root ganglion (DRG) sensory neurons after nerve injury, where its levels correlate with pain hypersensitivity in rodent neuropathic pain models, as well as in patients with chronic pain (Tegeder et al., 2006; Campbell et al., 2009; Kim et al., 2010; Lötsch et al., 2010; Heddini et al., 2012; Nasser and Møller, 2014; Belfer et al., 2015; Sadhu et al., 2018; Zheng et al., 2019). Targeting BH4 synthesis peripherally in injured sensory neurons represents a novel and potentially safe approach to combat certain neuropathic pain conditions (Latremoliere et al., 2015; Fujita et al., 2019). Since the early 1990s, the focus of drug discovery has been driven by target-based screening, using structural modeling and in silico computational analyses to design and screen small molecules using simple target-dependent assays, to find those that enter and block a certain enzymatic domain or binding motif (Sams-Dodd, 2005; Swinney, 2013). However, before this target-based approach, phenotypic screening was prevalent, often with limited information on the underlying mechanisms involved in the disease in question, and in which no targets were known or identified. Indeed, the success of these phenotypic screens over the more precision-targeted screens is revealed by the fact that the majority of first-in-class drugs actually came from phenotypic screens (Swinney and Anthony, 2011). Here, we describe a phenotypic screening platform using primary DRG sensory neurons from transgenic Gch1-GFP reporter mice that allowed us to monitor the effects of chemical libraries on the regulation of Gch1 expression in individual DRG neurons after axonal injury (axotomy). Using this platform, we provided not only novel insights into the biology of GCH1 expression and BH4 synthesis in injured sensory neurons, but also identified FDA-approved compounds with existing safety and pharmacokinetic profiles, which could potentially be repurposed to block the GCH1/BH4 pathway in neuropathic pain (Cronin et al., 2022). This protocol can be adapted to monitor the effects of compounds on the expression of multiple algesic genes in primary DRG cultures from transgenic mice or on human stem cell–derived sensory neurons.