Geoffery Burnstock’s influence on the evolution of P2X3 receptor pharmacology

Introduction

I first met Geoffery Burnstock in 1985 while working as a young post-doctoral fellow for Michael Williams at CIBA-Geigy Corporation. During that time, we were researching the role of adenosine in stroke and cardiovascular disease. These research efforts crystallized into a drug discovery program aimed at developing selective adenosine receptor agonists as cardioprotective agents. The first potent and selective A_2A receptor agonist, CGS-21680, was discovered as part of this effort [1]. This high-affinity agonist was successfully radiolabeled [2] and quantitative receptor autoradiography studies demonstrated that A_2A receptors were discretely localized in the rat basal ganglia [3]. These data were considered controversial at the time since other radioligand binding studies with less selective ligands had indicated a much broader expression of the A_2A receptor in the central nervous system [4]. During my first discussions with Geoff, I had the opportunity to share some of these early data. Needless to say, that as a newcomer to the field, I was a bit nervous about discussing our work with such a pioneering scientist. I remember Geoff’s supportive enthusiasm for our new data and his encouragement not to let existing dogma hinder our exploration into this area of purine pharmacology. His open-minded approach and unfettered excitement for new ideas has served as an inspirational guidepost throughout my research career.

Shortly after I joined the Neuroscience Discovery team at Abbott Laboratories in 1995, a new drug discovery effort targeting novel analgesic mechanisms as potential non-opioid interventions for chronic neuropathic and inflammatory pain was initiated. During an internal scientific research governance meeting, a photomicrograph from Geoff’s UCL team showing the discrete expression of P2X3 mRNA selectively localized on peripherin-positive small diameter sensory nerves was shared ahead its publication [5]. Geoff’s willingness to share these exciting data indicating a potential specific role for a single member of the emerging P2X receptor family in nociceptive neurotransmission provided sufficient evidence to initiate a specific drug discovery research effort on the nociceptive role of P2X3 receptors. In retrospect, the notion that highly selective expression of individual members of the emerging purine receptor superfamilies (e.g., A_2A receptors in brain and P2X3 receptors on peripheral nerves) could drive physiologically relevant neuromodulation is striking. Furthermore, it served as the scientific foundation of much of my drug discovery research activities for the better part of two decades. Below is a brief overview of the historical development P2X3 receptor pharmacology, much of which was directly driven by Geoff’s interactions with a wide variety of academic and industrial purinergic investigators over the years. Specific questions and insights provided by Geoff on our work are highlighted to illustrate the significant impact Geoff had on our work.

P2 receptor discovery

Based on seminal experiments in guinea pig taenia coli in the 1960s, Burnstock and colleagues discovered non-adrenergic, non-cholinergic (NANC) neurotransmission [6, 7]. Subsequently, these investigators determined that ATP could satisfy the criteria as the neurotransmitter underlying NANC [8]. The concept of purinergic neurotransmission was more formally described in the 1972 Pharmacological Reviews paper [9] and was built upon other key finding of Drury and Szent-Györgyi [10] and Holton [11]. Burnstock further differentiated purinergic responses into adenosine-based (P1 receptors) and ATP-based P2 receptors [12] using the existing pharmacological knowledge. This classification scheme for the P2 receptors was further refined in 1985, again based on analysis of agonist and antagonist pharmacology [13]. However, the notion that ATP could function as a discrete neurotransmitter was met with much skepticism over the next two decades largely because of the ubiquitous and somewhat diffuse involvement of ATP and its many rapid metabolic degradation products in virtually all aspects of cellular physiology [6, 14].

At the turn of the twenty-first century, several key developments occurred that helped clarify our understanding of the complex contributions of purines in both homeostatic and pathological situations such as persistent pain [15–18]. The emergence of molecular biology enabled cloning and characterization of individual receptors that would eventually be classified as the distinct ADO-sensitive (P1) and ATP-sensitive (P2) receptors super families [6, 12, 18, 19]. Additionally, the selective localization of P2X3 receptors on peripheral sensory neurons [5, 20] aligned nicely with an emerging concept of peripheral sensitization via aberrant functioning of ion channels and G protein-coupled receptors as a mechanism underlying chronic non-adaptive nociception [21–24]. The realization that P1 and P2 receptors comprised a wide diversity of G protein-coupled receptors and ligand-gated ion channels also provided a new framework for understanding the complexities of purinergic signaling via both direct and indirect (metabolic) actions [16, 18, 19]. Extracellular ATP availability could arise via co-transmission or via cell lysis [25–27], and the actions of individual purines were tightly regulated by membrane-bound and soluble ecto-nucleotidases [28–30]. The ability of ATP to evoke ectopic neuronal hypersensitivity may not be solely dependent on intrinsic P2X3 or P2X2/3 receptor activation since functional interactions with other P2 receptors including P2Y₁ and P2Y₂, other ligand-gated ion channels (transient receptor potential, TRPV1), and multiple intracellular signaling pathways have been reported [31].

P2X3 receptor ligands

All P2X3 receptor agonists are ATP derivatives that have differential efficacies at individual P2X receptors [32]. Nucleotide-derived agonists can also negatively modulate the effects of ATP. The agonist α,β-meATP can attenuate fast-desensitizing P2X receptor responses via rapid receptor desensitization [7, 33–35]. Diadenosine polyphosphates (Ap_nA) are endogenous modulators that can activate P2 receptors [32]. Selective P2X3 receptor desensitization by diadenosine polyphosphates can occur in the absence of detectable agonist activity demonstrating the complex role of these molecules in purine receptor pharmacology [33, 35].

Burnstock identified the alkaloid adrenergic receptor antagonist quinidine as one of the first P2 receptor antagonists [9]. Suramin, PPADS, and dyes including reactive blue-2, Evans blue, and Cibacron blue are low potency and nonselective P2 receptor antagonists [32]. Interestingly, several of these dyes also act as allosteric potentiators of P2X₁ and P2X3 receptors in vitro [32, 35, 36]. Cibacron blue can function as an allosteric modulator of P2X3 receptor activation [36]. This dye can facilitate the functional recovery of desensitized P2X3 receptors and enhances the pronociceptive effects of peripherally administered P2X agonists and formalin [36, 37]. Diinosine polyphosphates (e.g., P¹P⁵-Di-[inosine-5′]pentaphosphate) are also effective blockers of fast-desensitizing P2X3 receptors [32, 35].

The cloning and systematic pharmacological characterization of individual P2X receptor subtypes has led to an improved understanding of receptor selectivity for P2X3 receptor antagonists [38]. For instance, BzATP, a compound that blocks P2X₇ receptors with micromolar affinity, has low nanomolar affinity for blocking P2X₁ and P2X3 receptors [38]. Another ATP derivative, TNP-ATP (Fig. 1), potently blocks P2X₁, P2X3, and P2X2/3 receptors [39, 40]. Detailed kinetic studies demonstrated that TNP-ATP competitively blocks the non-desensitizing P2X2/3 receptor and non-desensitizing chimeric P2X3 receptors [41, 42]. TNP-ATP is rapidly metabolized in vivo; however, local administration has been shown to block the excitatory effects of intradermal ATP [37, 43].

Fig. 1.

Chemical structures of potent (nM) P2X3 receptor antagonists

A-317491 (Fig. 1) was the first described potent P2X3/P2X2/3 receptor-selective antagonist [44]. A-317491 is a competitive and reversible P2X3 antagonist that has good systemic bioavailability; however, it is poorly water soluble and has limited oral bioavailability [45]. Subsequently, a diaminopyrimidine class of P2X3 antagonists was discovered that have improved physicochemical properties compared with A-317491. [46–48]. This class of P2X3 antagonists bind to an allosteric site on the channel [49]. A lead antagonist, gefapixant (AF-219, MK-7264, Fig. 1), is a potent, reversible, peripherally restricted noncompetitive antagonist of P2X3 and P2X2/3 receptors [50]. In experimental pain, urological and respiratory models, gefapixant, and structurally related compounds block P2X3 receptor-dependent action potentials on afferent sensory neurons [50, 51].

Gefapixant has been studied in clinical trials for osteoarthritic joint pain, bladder pain syndrome/interstitial cystitis, and refractory cough [51–53, https://www.businesswire.com/news/home/20200317005183/en/Merck-Announces-Top-Line-Results-Phase-3-Trials]. In phase II and III trials for cough, gefapixant produced dose-dependent reductions in awake cough frequency following 12 weeks of treatment in patients with refractory chronic cough or unexplained cough [52]. However, perturbations in taste sensitivity were reported as a consistent tolerability finding in gefapixant-treated patients [52]. The available data indicates that this adverse event may be dose-dependent [52]. Gefapixant-mediated dysgeusia is likely due to block of P2X2/3 receptors based on the known expression of P2X3 and P2X2/3 receptors on taste buds [53], and altered taste sensitivity has been reported in double knockout mice lacking both P2X₂ and P2X3 receptors [54]. First generation non-nucleoside P2X3 antagonists including gefapixant block P2X3 and P2X2/3 receptors with similar potencies [55]. However, some newer P2X3 antagonists including the imidazo-pyridine, BLU-5937 (Fig. 1), have been reported to have stereoselective potency to block P2X3 receptors as compared with P2X2/3 receptors [54]. In a guinea pig cough model, BLU-5937 produced significant antitussive activity with little or no effect on taste sensitivity. BLU-5937 is currently in a proof of concept trial for chronic cough [54]. The individual contributions of specific P2X3 or P2X2/3 receptor block underlying altered taste sensitivity are not known. Furthermore, relative potencies of antagonist activity at homomeric P2X3 and heteromeric P2X2/3 receptors are difficult to interpret due to their vastly different desensitization profiles and multiple recognition sites [41, 51].

Nociceptive P2X3 receptor physiology and pharmacology

ATP inactivation by soluble and membrane-bound enzymes generates ADP, AMP, and ADO forming a purinergic cascade of extracellular messengers, each having distinct purinergic receptor functional profiles [14]. For example, ADO produces neuroprotective, sedative, and anticonvulsant actions, whereas ATP can mediate excitatory neurotransmission on central and peripheral nerve cells [14, 55].

The rat P2X3 receptor was originally cloned from rat dorsal root ganglion neurons [5, 20]. Both message and functional studies demonstrated the expression of homomeric fast-desensitizing receptors (P2X3) and more slowly desensitizing heteromeric (P2X2/3) receptors [56, 57]. Immunohistochemical studies showed that P2X3 receptors are expressed at central and peripheral terminals, as well as cell bodies, of small diameter sensory neurons [58–61]. With regard to pain sensitivity, ATP, released from damaged cells, initiates a nociceptive signal [27]. Presynaptic P2X3 and P2X2/3 receptors located at the first central synapse of primary afferent neurons can also enhance neurotransmission [59, 62].

The neuroanatomical distribution of P2X3 and P2X2/3 receptors is functionally important. Local exogenous administration of ATP elicits pain in humans both under normal [63] and inflammatory states [64, 65]. Similarly, intradermal administration of P2X receptor agonists, such as ATP and α,β-methyleneATP (α,β-meATP), produce acute dose-dependent nociceptive responses that are similar in magnitude to the acute phase of the formalin test [33, 36, 66–69]. The potency and effectiveness of locally administered P2X receptor agonists to stimulate nociceptive responses are increased in situations of peripheral sensitization induced by inflammation [68, 70]. Damaged rat sciatic nerve fibers also show an ectopic sensitivity to ATP [71].

The nociceptive effects of locally administered P2X receptor agonists measured in preclinical models can be attenuated by systemically administered morphine [33] and by the local co-administration of putative P2 receptor antagonists [70]. The intrathecal administration of P2X receptor antagonists also produce antinociception in a wide variety of pain models including acute, inflammatory, and neuropathic pain assays [72–75]. In studies using the P2X3 selective antagonist, A-317491, inflammatory hyperalgesia, and neuropathic mechanical allodynia were reduced in rodent experimental pain models [44]. The less active R-enantiomer of A-317491, A-317334, was inactive in the pain models providing further evidence for pharmacological specificity [44].

The regulation of P2X3 receptor expression in the context of neuronal trauma is complex. DRG P2X3 receptor mRNA is reduced in injured peripheral nerves and increased in adjacent uninjured nerves following nerve ligation [76, 77]. There are also reports of increased P2X3 expression in dorsal root ganglion small and medium diameter neurons in models of inflammatory neuropathic pain [78]. In a peripheral nerve injury model, spinal cord P2X3 immunoreactivity is increased on the side ipsilateral to nerve injury [71]. However, in a neurogenic-driven allodynia model, overall P2X3 receptor expression was decreased in dorsal root ganglion neurons ipsilateral to the nerve injury due to a selective decrease in fast-desensitizing homomeric P2X3 receptor expression [79]. In this model, the expression or function of P2X2/3 receptors on larger diameter DRG neurons remained unchanged [79]. Together, these receptor regulation studies provide some evidence for direct roles of homomeric and heteromeric P2X3 containing channels in mediating acute and chronic pain. As noted earlier, there are also many indirect intracellular signaling mechanisms linked to P2X3 receptor activation that likely modulate receptor desensitization kinetics and overall nociceptive neurotransmission [23, 24, 31].

Data derived from the study of transgenic knockout mice provided evidence that P2X3 receptors mediate the sensation of some types of peripherally mediated pain [80, 81]. However, interpretations of gene disruption studies have historically been limited by the selection of in vivo assays as well as the potential of confounding factors including mouse strain, gene compensatory changes, and environmental influences [80–82]. In early reports, acute noxious thermal and mechanical sensitivity was not significantly different between P2X3 receptor (−/−) and P2X3 (+/+) mice [81], yet P2X3 receptor (−/−) mice did show reduced sensitivity to peripherally applied ATP and formalin as compared with wild-type mice [80, 81]. P2X3 receptor (−/−) was reported to exhibit a hyperalgesic response to subchronic inflammation and altered neural processing of mild “warmth” thermal stimuli [81]. P2X3 knockout mice have also exhibited altered ambient thermal preferences [83]. Transgenic mice lacking both P2X₂ and P2X3 receptors also show alterations in taste sensitivity to bitter and sweet substances [53].

Transient gene knockdown studies using P2X3 receptor antisense oligonucleotides provided further insights on effects of short-term P2X3 receptor disruption [84, 85]. P2X3 antisense treatment decreases sensitivity to the nociceptive effects of locally administered ATP and formalin in rats [86.87]. P2X3 antisense treatment attenuated thermal hyperalgesic responses in an inflammatory pain model and reversed tactile allodynia in multiple models of neuropathic pain [84, 85].

Summary and commentary

The scientific advances in the field of purinergic pharmacology over the last three decades would not have been possible without Geoff Burnstock’s curiosity, enthusiasm, persistence, and collegiality. His willingness to work with all scientists interested in purine biology and his encouragement of early career scientists are especially noteworthy. While these attributes led to open skepticism regarding many of his ideas over the course of his career, the development of P2X3 pharmacology and the advancement of a receptor-selective antagonist into advanced clinical trials clearly validates his vision regarding the fundamental role of ATP in neurotransmission. From a drug discovery perspective, his encouragement and collaboration with pharmacologists at Roche and Abbott Laboratories during the late 1990s and early 2000s were invaluable. He was a consistent champion of pharmacological data especially in the face of ambiguous findings from the early investigation of gene disrupted mice [80, 81]. A-317491 was discovered before the mouse knockout phenotypes were known which caused our group to question the apparent specificity of this novel antagonist. It was Geoff’s perspective on the complexities of transgenic mouse studies that led the Abbott team to undertake the P2X3 antisense studies [84] which confirmed our emerging pharmacological data with A-317491 [44]. Ultimately, Geoff’s collaboration with the Roche/Afferent team led to the discovery of the orally bioavailable P2X3 antagonist, AF-219 (gefapixant). The advancement of this important antagonist into multiple and diverse proof of concept clinical trials [51, 52, 86] for human diseases highlights the key role of purinergic signaling in aberrant peripheral sensory nerve sensitivity. The focus of this brief review has been based primarily on Geoff’s influences on our work regarding purinergic modulation of nociceptive physiology. The emerging clinical data for gefapixant offers key lessons on the exploratory nature of drug discovery and development for novel mechanistic interventions. Importantly, the optimization of P2X3 receptor-selective antagonists as pharmacological tools have also been instrumental in the study and validation of P2X receptor structure and function [55, 87].

Compliance with ethical standards

Conflicts of interest

Michael F. Jarvis declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.