Pharmacological characterization of a structural hybrid P2X7R antagonist using ATP and LL-37

Abstract

Antagonists of the P2X7 receptor (P2X7R) have the potential to treat diseases where neuroinflammation is present such as depression, chronic pain and Alzheimer's disease. We recently developed a structural hybrid (C1; 1-((adamantan-1-yl)methyl)-2-cyano-3-(quinolin-5-yl)guanidine) of a purported competitive P2X7R antagonist (C2; 2-cyano-1-((1S)-1-phenylethyl)-3-(quinolin-5-yl)guanidine) and a likely negative allosteric modulator (NAM) of the P2X7R (C3; N-((adamantan-1-yl)methyl)-2-chloro-5-methoxybenzamide). Here we aimed to pharmacologically characterize C1, to gain insights into how select structural components impact antagonist interaction with the P2X7R. A second aim was to examine the role of the peptide LL-37, an apparent activator of the P2X7R, and compare the ability of multiple P2X7R antagonists to block its effects. Compounds 1, 2 and 3 were characterised using washout, Schild and receptor protection studies, all using dye uptake assays in HEK293 cells expressing the P2X7R. LL-37 was examined in the same HEK293 cells and THP-1 monocytes. Compounds 2 and 3 acted as a BzATP-competitive antagonist and NAM of the P2X7R respectively. Compound 1 was a slowly reversible NAM of the P2X7R suggesting the incorporation of an appropriately positioned adamantane promotes binding to the allosteric site of the P2X7R. LL-37 was shown to potentiate the ability of ATP to induce dye uptake at low concentrations (1–3 μg mL^-1) or induce dye uptake alone at higher concentrations (10–20 μg mL^-1). None of the P2X7R antagonists studied were able to block LL-37-induced dye uptake bringing in to question the ability of current P2X7R antagonists to inhibit the inflammatory action of LL-37 in vivo.

Introduction

The P2X7 receptor (P2X7R) is an ATP-gated cation channel (North, 2002). Activation leads to the processing and release of inflammatory mediators including interleukin-1β (Barberà-Cremades et al., 2012; Ferrari et al., 1997). Prolonged P2X7R stimulation produces a cytolytic pore, permeable to fluorescent dyes up to 900 Da (Pelegrín, 2011; Steinberg et al., 1987). P2X7R expression throughout the body is near ubiquitous with high expression seen on macrophages and microglia (Collo et al., 1997; Wiley et al., 2011). Given this, antagonists of the P2X7R are a potential treatment for diseases involving neuroinflammation (Bhattacharya and Biber, 2016; Skaper et al., 2010), such as chronic pain, Alzheimer's disease and depression (Heneka et al., 2015; Ji et al., 2014; Setiawan et al., 2015).

LL-37 is a 37 amino acid, human antimicrobial peptide (Elssner et al., 2004). A receptor-mediated action of LL-37 has been identified, as the peptide was revealed to be an activator of P2X7R-mediated responses (Elssner et al., 2004).

Multiple series of potent, drug-like, P2X7R antagonists have been developed including the adamantyl benzamide, cyclic imide and aryl cyanoguanidine series (Alcaraz et al., 2003; Donnelly-Roberts et al., 2009). AstraZeneca's Compound 3 (Fig. 1), a member of the adamantyl benzamide series (pA₂ = 8.8), was originally discovered following hit-to-lead studies. There is no report about the mechanism of action of C3, despite the inclusion of pA₂ values suggesting Schild analysis was performed (Alcaraz et al., 2003). Compound 4 (3-(1-((3′-nitro-[1,1′-biphenyl]-4-yl)oxy)-4-(pyridin-4-yl)butan-2-yl)thiazolidine-2,4-dione; Fig. 1), a member of the cyclic imide series, is a negative allosteric modulator (NAM) (Alcaraz et al., 2003; Michel et al., 2009). Later work on another adamantyl benzamide P2X7R antagonist (Compound 5) suggests that this compound is also a NAM (Michel et al., 2009). The aryl cyanoguanidine C2 (Fig. 1) was originally reported to be BzATP-competitive (Donnelly-Roberts et al., 2009), potentially indicating orthosteric antagonism. We recently published a hybrid series of adamantyl cyanoguanidine P2X7R antagonists and produced a lead compound (C1, Fig. 1) with greater potency than compounds from either parent series (O'Brien-Brown et al., 2017).

Despite the existence of multiple series of P2X7R antagonists, the lack of pharmacological characterization of these series has been noted (Michel et al., 2008). This deficit has been rectified somewhat, partially due to the development of receptor protection studies at the P2X7R which allow characterization of slowly reversible orthosteric antagonists and NAMs which prove difficult to characterize via Schild assays (Michel et al., 2006, 2008). Nonetheless pharmacological characterization needs to be completed for each new series produced, so that their suitability as tool compounds or even candidates for drug development can be evaluated. Further, no drug-like lead compound has been characterized in terms of its ability to inhibit LL-37-induced responses despite the recognition that LL-37 has a receptor-mediated action at the P2X7R.

Here we engaged in pharmacological characterization of our lead compound, C1, aiming to determine its mechanism of action. Further, we aimed to confirm LL-37 as an activator of the P2X7R and examine the ability of multiple P2X7R antagonists to inhibit LL-37-induced pore formation.