Purinergic P2X7 receptor antagonists: Chemistry and fundamentals of biological screening

Bioorganic & Medicinal Chemistry 17 (2009) 4861–4865

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Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Perspective

Purinergic P2X7 receptor antagonists: Chemistry and fundamentals of biological screening Hendra Gunosewoyo a,b, Mark J. Coster c, Maxwell R. Bennett b, Michael Kassiou a,b,d,* a

School of Chemistry, University of Sydney, NSW 2006, Australia Brain and Mind Research Institute, 100 Mallett St, Camperdown, Sydney, NSW 2050, Australia c Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Qld 4111, Australia d Discipline of Medical Radiation Sciences, University of Sydney, NSW 2006, Australia b

a r t i c l e

i n f o

Article history: Received 16 March 2009 Revised 5 May 2009 Accepted 6 May 2009 Available online 12 June 2009

a b s t r a c t The purinergic P2X7 receptor is a unique member of the ATP-gated P2X family. This receptor has been implicated in numerous diseases and many structurally diverse ligands have been discovered via high throughput screening. This perspective will attempt to highlight some of the most recent key findings in both the biology and chemistry. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: P2X7 antagonists Cytokine release Rational drug design LL-37

1. Introduction

2. P2X7R Receptor biology

The purinergic P2X7 receptor (P2X7R) is the most unusual member of the P2X receptor family in terms of its structure and function. Since the isolation and characterization of the P2X7R from rats, mice, and humans in the late 1990s, there has been increasing interest in delineating physiological aspects of P2X7R function. Of particular interest is elucidating its role in neuroinflammation, neurodegenerative diseases, depression and modulation of pain states. Although there have been recent advances in our understanding of the P2X7R in these conditions, this will not be covered in this article. High throughput screening methodology resulted in the initial identification of P2X7R antagonists.1,2 In recent years, there has been an increase in the available chemical diversity of P2X7R antagonists as evidenced by the number of patents filed by pharmaceutical companies and academic units over the past decade. A number of reviews on the P2X7R have been published.3–8 This perspective aims to highlight some of the most recent key findings in both the biology and chemistry of this rapidly growing area of research.

The P2X7R is an ion channel activated by extracellular ATP.9 Seven subtypes of P2X receptors (P2X1–7) have been currently identified in rodents and humans. 20 (30 )-O-(4-Benzoylbenzoyl)adenosine 50 -triphosphate (BzATP) 1 (Fig. 1) is the most potent synthetic ATPderived agonist acting at the P2X7R and therefore is utilized in almost all functional assays to date. The guinea pig P2X7R orthologue has also been successfully cloned and characterized.10 Interestingly, at this orthologue, BzATP was found to be a partial agonist compared to ATP and depending on the buffer composition, it surprisingly acted as an antagonist, exemplifying the very existence of species differences amongst the P2X7R orthologues.8 The P2X7R is ubiquitously found in a variety of cell types, most notably those of haematopoietic origin, such as mast cells, macrophages, and lymphocytes, as well as glial cells, including microglia and astrocytes. It possesses a cytoplasmic, 240-amino acid residue carboxy terminal tail that is believed to be necessary for its bifunctionality as a selective ion channel assembled from P2X7 subunits, or, as a non-selective pore.11 There are two hypotheses explaining P2X7-associated pore formation;9 in both scenarios brief activation of the receptor with ATP leads to opening of the ion channel by conformational changes within the P2X7 structure. It was initially thought that the pore formation occurs as a result of dilation of the ion channel. This idea is supported by the progressive nature of channel-to-pore formation and its occurrence in almost all cells expressing the receptor. Alternatively, prolonged activation of the

* Corresponding author. Tel.: +61 2 9351 0849; fax: +61 2 9351 0852. E-mail addresses: [email protected], [email protected] (M. Kassiou). 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2009.05.083

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P2X7 receptor complex leads to activation of intracellular machineries that signals the opening of P2X7-independent pore-forming proteins to allow entry of large (up to molecular weights of 900 Da) cationic species. Supporting the hypothesis that the pore is a separate entity from the channel is the key findings that pannexin-1 showed protein–protein interaction with the P2X7R and that carbenoxolone, small interfering RNA (siRNA) directed against pannexin-1, and a pannexin-1 mimetic peptide all blocked the initial phase of the pore formation without interfering with ion flux.12–15 However, there is now increasing evidence to contest the role of pannexin-1 in P2X7R pore formation.16,17 While it was thought that ATP activation of the P2X7R signals the opening of pannexin-1 channels and subsequent entry of large cations or dye uptake, it was found that inhibition of pannexin-1 currents by some gap junction blockers (with the exception of DIDS) does not block P2X7R currents. Furthermore, ATP was found to cause a rapid and reversible inhibition of pannexin-1 currents at a dose needed for P2X7R activation (about 1 mM in HEK293 cells). Taken together, all these results demonstrate the complexities of the P2X7 channel-to-pore formation and that further identification of signaling molecules linked to the P2X7 channel is still needed if the ‘pore as a separate entity’ hypothesis were to be correct. In 2004, Elssner et al. reported the ability of an endogenous ligand LL-37,18 a potent antimicrobial peptide produced predominantly by neutrophils19 and epithelial cells, to induce interleukin-1b (IL-1b) release via direct activation of the P2X7R and that ATP may not be needed for this response.20 Recently, Tomasinsig et al. studied this endogenous P2X7R agonist peptide18–20 derived from the human cathelicidin protein hCAP-18 in further detail.21 The key results were: (i) LL-37 alone promotes Ca2+ influx mediated by human P2X7R (hP2X7R), (ii) LL-37 potentiates the Ca2+ influx response obtained with BzATP alone, (iii) LL-37 induces ethidium bromide uptake and potentiates the BzATP-stimulated plasma membrane permeabilisation, and lastly, (iv) LL-37 promotes pore formation in carboxy tail-truncated hP2X7R. This last finding is extraordinary as it is widely believed that the carboxy terminal tail is necessary for the unique pore-forming characteristic of the P2X7R11 and has been shown to serve as a regulatory module for the P2X7R channel activity.22 The authors reasoned that the unique, strong helix-forming propensity property of LL-37 is crucial for the P2X7 activity. To date, there is no structural knowledge available to show the exact mode of interaction between LL37 and P2X7R as well as other receptors. The obvious implication from this study is that the carboxy terminal region of P2X7R is not absolutely required for the binding of LL-37 to the receptor complex. In light of the ‘pore as a separate entity’ hypothesis of the channel-to-pore formation, LL-37 is suggested to possess the ability to replace the carboxy tail as an alternative linker to the unidentified machineries that signal the pore opening.21 Moreover, suppression of pannexin-1 expression by siRNA results in no decrease of dye uptake, further indicating the unlikely involvement of pannexin-1 in the P2X7R pore formation. It has been long-debated whether the activation of P2X7R by endogenous ATP which is required in millimolar concentration is physiologically relevant. The purinergic P2X7R has a characteristically low affinity of ATP compared to other P2X subtypes. While this ATP concentration corresponds to the cytosolic concentration in vivo, it is possible that other unidentified, endogenous molecules may be involved in the initial activation process of the receptor. In this context, LL-37 represents a candidate endogenous molecule that results in the activation of the receptor, that is, channel-to-pore formation at low micromolar concentrations which is physiologically relevant. It could be further suggested that LL-37 in combination with ATP results in the full activation of the P2X7R in disease states.

The screening of novel P2X7R antagonists to date has solely utilized ATP or its synthetic derivative BzATP for activation of the receptor. In light of the identification of LL-37 as a highly potent endogenous agonist which appears to be more physiologically relevant, it is suggestive that screening of P2X7R-active molecules should utilize LL-37 in place of ATP or its synthetic derivatives. This new screening paradigm may provide a more accurate characterization of the potency of these novel P2X7R molecules. It is worthwhile to note that there are other reported compounds that potentiate P2X7-associated responses, such as tenidap,23 bromoenol lactone,24 and polymyxin B.25,26 However, the exact mechanism of action of these compounds is largely unknown. 3. Molecular probes of The P2X7R: an update No structure-based drug design based on the knowledge of binding sites within the P2X7R has been undertaken. This is due to the fact that no crystal structure or suitable homology model27–29 have been reported for this receptor. The new classes of P2X7R antagonists developed to date are derived almost exclusively from high throughput screening methods using BzATP-induced cell permeabilisation as the biological basis. This section serves as an update for the previously reviewed available P2X7-active molecules.8 3.1. Aryl hydrazides High throughput screening of the Abbott Laboratories’ compound library yielded aryl hydrazides30 as a novel class of P2X7R antagonists. Compound 2 (Fig. 2) possesses pIC50 values of 7.99 and 7.35 at human and rat P2X7R, respectively when tested for inhibition of calcium flux. Intraperitonial administration of this molecule at 20 lmol/kg dose in an in vivo model of zymosan-induced peritonitis displayed more than 60% reduction of IL-1b production. This compound has also been efficacious in the spinal nerve ligation model of neuropathic pain and had no interaction with other P2 receptors, such as P2X3, P2X4 and P2Y2 at 10 lM concentration. Furthermore, compound 3 (Fig. 2) is found to be inactive in a variety of 70 receptors, enzymes and ion channels at 10 lM concentration. 3.2. Cyanoguanidines The cyanoguanidine scaffolds for use in development of P2X7Rselective antagonists were first reported by the Abbott Laboratories. The particular highlight of this class of compounds is the minimal species differences across rodents and human. In 2006, compound 4 (A-740003), with IC50 values (calcium flux inhibition) of 40 and 18 nM for human and rat P2X7R, respectively, was found to reduce neuropathic pain in a rat model in a dose-dependent manner (Fig. 3).31 The same compound was also assessed for inhibition of IL-1b release (IC50 156 nM) and pore formation (IC50 92 nM) at the hP2X7R. In addition, there is hardly any significant

NH2 N

N

N N O O O HO P O P O P O O OH OH OH 3' 2' (H)RO OH(R)

O R=

mixture of 2' and 3' esters O

1: BzATP Figure 1. Structure of BzATP: the most potent synthetic ATP-derived P2X7 agonist.

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O

H N

N

Cl

H N

N H 2

O

Cl Cl

N H

Cl

N N

N

N N

Cl

Cl

N

Cl

N

HN

HN

HN N

N

3

N N N

N

N

N

Figure 2. Aryl hydrazides.

9

10

11

Figure 4. Triazoles.

OCH 3 OCH3 H N

N

H N

NC

H N

N

OCH3 O

N

N

H N

OCH3 NC

N 5

4: A-740003 Cl

O R N

N

H N

N

NC

O

N

N H

3H

N

NC

N

6: R = H 7: R = Me

H N

H N N

8: [3H]A-804598 Figure 3. Cyanoguanidines.

activity at other P2 receptors and neurotransmitters at 10 lM concentration. In 2008, the piperazine-incorporated cyanoguanidine 5 was reported to possess IC50 values of 30 and 59 nM at rat and human P2X7R, respectively.32 The selectivity for P2X7R against other P2 receptors, such as P2X2/3, P2X3, P2X4 and P2Y2 at 10 lM concentration is still maintained. Subsequent studies focused on the optimization of these cyanoguanidines for improved potency in human whole blood assay.33 Compound 6 has IC50 values of 57, 40 and 121 nM at the rP2X7R, hP2X7R and whole blood assay, respectively. Installment of a methyl group at the quinoline ring (7, Fig. 3) was found to impart greater microsomal stability in the whole blood assay. The most recent cyanoguanidine derivative reported in the literature is compound 8 (Fig. 3) with IC50 of around 10 nM at all rat, human and mouse P2X7R.34 Tritium-labeled 8 has been used as a radioligand in rat P2X7R binding studies with an excellent selectivity profile over other P2 receptors, cell surface receptors or ion channels. 3.3. Triazoles Triazoles 9–11 represent some of the Abbott Laboratories’ most potent analogues from this series with pIC50 values >7.50 at both human and rat P2X7R in Ca2+ flux inhibition assay (see Fig. 4).35 Compounds 9 and 10 were also found to be inactive at other P2 receptors, such as P2X1, P2X2, P2X2/3, P2X4, P2Y1 and P2Y2 at 10 lM concentration. Replacement of the triazole core with other azoles has been reported.36 It was observed that the position of nitrogens in the core did not dictate activity; several tetrazoles and triazoles with different connectivities were assayed and compared. There was, however, a linear relationship between the overall electron density of the core and P2X7R potency in the order: tetrazole > triazole > imidazole > pyrazole. The triazoles were reported to possess similar potency to the tetrazole analogues with improved physicochemical properties and one example from this class has also been evaluated in a rat model of neuropathic pain.

3.4. Polycyclic benzamides The adamantyl benzamides as P2X7R antagonists were first reported by Astra Zeneca in 2003.37 The first series of these adamantyl benzamides, however, only possessed activity at the hP2X7R and not at the rodent orthologues. Subsequent studies by Astra Zeneca38 reported that substitution at the 5-position of the aromatic ring (12 and 13) introduced activity at the both the rat and human P2X7R (Fig. 5). Interestingly, it was possible to achieve species selectivity upon further substitution at the aromatic ring. Compounds 12 (pA2 value of 7.8 at the rP2X7R) and 13 are selective for rat over human P2X7R (pIC50 <6) and both compounds have excellent selectivity profiles amongst the P2X receptors (P2X1–5) as well as other common receptors.38 More recently, compound 14 has been studied in both acute and chronic rat models of pain and inflammation with promising results for treatment of rheumatoid arthritis.39 In our own laboratory, we also identified cubyl benzamide 15 as a novel P2X7R antagonist.40 Systematic modifications of the aromatic and polycyclic sites of these scaffolds would be likely to result in potent, selective P2X7R antagonist. 3.5. Natural products scaffolds Stylissadines A and B (16 and 17, Fig. 6) were discovered from high throughput screening of natural product extracts library in Australia.41 The biological screen used was the inhibition of BzATP-mediated pore formation in human THP-1 cells. The IC50 values for stylissadines A and B are 700 nM and 1.8 lM, respectively. It is interesting to note that massadine 18 (Fig. 6),42 the monomer of the styllisadines A and B, is a non-selective antagonist of the BzATP-mediated pore formation as it shows both activity at the P2X7R and hemeolysin specificity assay. Despite the low potency, the tetrameric pyrrole–imidazole alkaloids stylissadines A and B are the first reported natural product-derived P2X7R antagonist. More recently, some P2X7-active protoberberine alkaloids have been discovered from high throughput screening of the Korea Chemical Bank.43 Compound 19 (Fig. 7) was found to be the most potent analogue from the SAR studies and possesses IC50 values comparable to that of KN-62 (0.3 lM) when screened at the hP2X7R for inhibition of pore formation and IL-1b release.

NH O O

N 5

6

O

2

R

12: R = Me 13: R = Cl

NH2

4

1

N H

N

3

H N

F

H N O

Cl

14 Figure 5. Polycyclic benzamides.

O

15

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NH2 HN O

4 CF3COO

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NH2

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16: Stylissadine A

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NH OH H

NH

HO NH2

HO HN

HN

NH O NH

NH2 17: Stylissadine B

Br Br Br

H N

NH

H HO HN

O

N H

2 CF3 COO

Br NH2 HN O HN H2N

NH OH H NH

OH

O HN NH

Br Br

H N O

Br

N H

Br

18: Massadine

Figure 6. Natural products scaffolds.

O O

N

OMe OMe

I O O

NO2

19 Figure 7. Protoberberine.

4. Conclusions A number of new classes of P2X7R antagonists have been reported by pharmaceutical companies and academia in recent years. This is attributed to the increasing recognition of P2X7R involvement in a wide variety of disease states. Concurrently, the lack of P2X7R structural information, a suitable homology model, determination of suitable pharmacophores and characterization of binding site(s) continues to hamper our understanding of P2X7R function in normal and disease states. Direct labeling studies have shown that the P2X7R antagonists are non-competitive at the ATP binding site. Furthermore, not all P2X7R antagonists bind at the same site which may have implications in therapeutic efficacy.44 At present, the exact mechanism of channel-to-pore transition remains to be elucidated. However, LL-37 represents an endogenous agonist with higher affinity to P2X7R than ATP and that it is likely to have a role in the P2X7 activation in a physiologically relevant manner. Taken together the co-application of ATP and LL-37 for full activation of the receptor studies and screening of new P2X7R antagonists should be considered as the model of choice. Acknowledgment H.G. would like to acknowledge CSIRO Postgraduate Top-up Scholarship. References and notes 1. Guile, S. D.; Alcaraz, L.; Birkinshaw, T. N.; Bowers, K. C.; Ebden, M. R.; Furber, M.; Stocks, M. J. J. Med. Chem. ASAP 2009, 12, 23. 2. Carroll, W. A.; Donnelly-Roberts, D. L.; Jarvis, M. F. Purinergic Signalling 2009, 2009, 63. 3. Burnstock, G. Nat. Rev. Drug Disc. 2008, 7, 575. 4. Burnstock, G. J. Physiol. (Oxford, UK) 2008, 586, 3307. 5. Inoue, K. Cell. Mol. Life Sci. 2008, 65, 3074. 6. Matute, C. Mol. Neurobiol. 2008, 38, 123. 7. Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez-Cara, C.; Preti, D.; Borea, P. A.; Gessi, S. Expert Opin. Ther. Targets 2008, 12, 647. 8. Gunosewoyo, H.; Coster, M. J.; Kassiou, M. Curr. Med. Chem. 2007, 14, 1505. 9. North, R. A. Physiol. Rev. 2002, 82, 1013.

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