Immuno-Oncology Therapeutic Potential of Adenosine Receptors: Targeting the Purinergic Pathway

 

While the discovery of adenosine effect goes back more than a century, today there seems to be a real craze for the targeting of adenosine receptors (a.k.a ADORA) as potential targets for new drugs, whether small molecules or monoclonal antibodies.

 

But who are these receptors, what are they used for in the human body and where are they present?

Why is it interesting to target them and for which therapeutic indications?  

What are the real challenges of a pharmaceutical development targeting adenosine receptors?

And is there still room for new biotechnological developments?

 

Just an outline of the few dozen questions one might have about such transmembrane receptors of interest. And as the saying goes, if you want to know where you're going, look at where you came from. So let's go back in time a bit first.

 

The discovery of purinergic signaling: a complex mix of transmembrane receptors, enzymes and unique mediators

 

The story goes that it all began with the isolation of a particular complex, the muscle adenylic acid (AMP), by Gustav Embden and Margarete Zimmermann (1) as early as 1927 in their work on skeletal muscle, and simultaneously by Pohle's team (2) from brain samples.

 

In fact, it turns out that Bass and his collaborators had already isolated the substance from blood during their work in 1914 (3).

 

If you have the patience to investigate further the origins of the discovery of purines, you will find that in 1776, Carl Wilhelm Scheele, one of the greatest chemists of all time, isolated for the first time uric acid, a molecule resulting from the breakdown and excretion of purines (4).

 

Based on the discovery of adenylic acid, Druiy and Szent Gyorgyi then investigated further the molecule to finally discovered extracellular signalling roles of adenosine on the cardiac rhythm (5). This discovery led to the approval of the first adenosine receptor-related drug for human use: adenosine itself, for the life-threatening condition of paroxysmal tachycardia.

 

The concept of “purinergic nerves” was finally developed by Geoff Burnstock in 1972, establishing that adenosine triphosphate (ATP) can be considered to be a neurotransmitter.  The related transmembrane receptors of adenosine (ADO), ATP and adenosine diphosphate (ADP) were described and categorized by Burnstock in 1978 (6).

 

Adenosine is a very unique molecule, with the gift of ubiquity on Earth, and at a crossroad between several key metabolic pathways in cellular metabolism; it is a product from ATP degradation and is also a substrate for its synthesis. Adenosine is produced from extracellular ATP by the sequential activity of two adenosinergic ectoenzymes, CD39 and CD73. CD39 (ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1)) converts ATP and ADP to AMP, while CD73 (ecto-5-nucleotidase (NT5E)) converts AMP to adenosine.

 

Adenosine exerts its effects through purinergic receptors, also called purinoceptors. Purinergic receptors are divided into two major classes, P1 and P2, depending on the ligand with which they interact:

  • P2X receptors - first subclass of P2 family - are ion channels that specifically recognize adenosine triphosphate (ATP),
  • P2Y receptors – second subclass of P2 family - are G protein-coupled receptors (GPCRs) that can respond to ATP or adenosine diphosphate (ADP) binding,
  • P1 receptors are seven-(pass)-transmembrane domain receptors that only undergo a conformational change when adenosine or inosine binds to them.

New elements are constantly being added to the complex network of enzymes, receptors, transporters and mediators that makes up the purinergic signaling complex.

 

Targeting P1 transmembrane receptors family: activating or blocking adenosine receptors A1AR, A2AR, A2BR and A3AR

 

In 1989, in Belgium, Libert and Parmentier identified members of a new subfamily of receptors with a very short nonglycosylated amino-terminal extension and so discovered A1, A2A and A2B receptor subtypes (7). In 1992, on the other side of the Atlantic, Zhou et al. reported the functional characterization of a newly discovered adenosine receptor: the A3 subtype was identified (8).

 

Since adenosine is released from pre- and postsynaptic components as well as from glial cells, the first therapeutic application of adenosine receptor antagonists and agonists was naturally studied in central nervous system dysfunctions such as stroke during WWII (Stoner and Green, 1945), epilepsy (Chin et al, 1989), psychiatric disorders, drug addiction, neurodegenerative disorders and more recently sleep disorders.

 

But as the concentration of extracellular adenosine also increases dramatically in stressful situations, such as ischemia, Presta et al. demonstrated the in-vitro and in-vivo anti-angiogenic effects of purine analogues as early as 1999 (9). Therefore, the potential use of therapeutics targeting adenosine transmembrane receptors could be extended to all diseases with tissue disruption, hypoxia, ectonucleotidase expression, and inflammation, including auto-immune diseases, wound healing (10), retinopathies (11) and coronary artery disease (12). As well as cancer and its tumor microenvironment (13, 14).

 

Oncology Therapeutic interest of G Protein-Coupled Receptor Adenosine Receptor A1AR (ADORA1)

 

Located on 1q32.1 locus human chromosome, A1 receptors have a widespread distribution in the body with a high density in the hippocampus, cerebral cortex, and cerebellum. A1AR is a glycoprotein containing a single complex carbohydrate chain of 326 amino acids with a mass of approximately 36.7 kDa.

 

At cellular level, they are located before and after the synaptic gap. They control neurotransmitter release and the frequency of action potentials. A1 receptors are coupled to pertussis toxin sensitive G-proteins (Gi, Gq, and Go family) that alpha subunits lead to an inhibition of the activity of the enzyme adenylate cyclase. However, through beta/gamma subunits they activate potassium channels, inactivate voltage sensitive calcium channels and also induces phospholipase C (PLC)-β activation, thereby increasing inositol trisphosphate (IP3) and intracellular Ca2+ levels.

 

ADORA1 serves as an important oncoprotein and a promoter of cell proliferation through PI3K/AKT signaling pathway in hepatocellular carcinoma (15). Consequently agonist for A1AR should demonstrate reduction of tumor progression: it is the case of small molecule antagonist DPCPX in hepatocellular carcinoma.

 

Status of oncology drugs targeting the G protein-coupled receptor adenosine A2AR (ADORA2A)

 

Adenosine A2A receptors (A2ARs) gene is located on 22g11.2 locus in human chromosome. A2AAR is a protein of 412 amino acids in length and 45 kDa.

 

A2A receptors are distributed in the liver, heart, lung, immune systems (spleen, thymus,

leucocytes, and blood platelets), and dopamine enriched areas.

 

It is now clear that the highest density of A2A receptors is confined to a discrete brain region, the dorsal and ventral striatum. In this brain region, A2A receptors are particularly abundant in plasma membrane of dendrites and dendritic spines and less in axon and axon terminals and glial cells, and are mostly expressed in the cells that express the D2 dopamine receptors. Antagonistic membrane and functional interaction exists between the A2A and D2 receptors. Therefore, blockade of A2A receptors mimics the action of D2 agonists. In contrast to A1AR, A2A and A2B receptors are coupled with G proteins Gs or Gq and activate adenylyl cyclase or phospholipase C.

 

Adenosine promotes gastric cancer cell invasion and metastasis by interacting with A2aR to enhance PI3K-AKT-mTOR pathway signaling (16).

 

Several antagonist small molecules blocking A2AR have been developed including:

  • ZM-241385 from AstraZeneca, combined with an anti-CTLA4 monoclonal antibody, but stopped at preclinical stage due to poor bioavailability,
  • SCH-58261 from Schering-Plough, stopped at preclinical stage due to poor physicochemical and pharmacokinetic properties
  • MK-3814, a structural derivative of SCH-58261, used in combination with Merck anti-PD-1 monoclonal antibody pembrolizumab (Keytruda) in phase Ib/II study, and terminated because the data did not support the study endpoints.
  • SYN-115 (tozadenant) from Roche, licensed to Synosia Therapeutics (acquired by Biotie, itself acquired by Acorda) and finally licensed to UCB.
  • Imaradenant from AstraZeneca (initially developed by Sosei Heptares) and stopped due to safety or efficacy during clinical trials. 

And some are still in oncology clinical trials:

  • AZD4635 (imaradenant) from AstraZeneca, developed by Sosei Heptares
  • EOS-100850 (Inupadenant) from iTeos Therapeutics, currently tested in combination with its anti-TIGIT mAb (EOS-448)
  • Ciforadenant from Corvus Pharmaceuticals (initially developed by Vernalis), currently tested in combination with Roche anti-PDL1 mAb atezolizumab
  • PBF-509 from Palobiofarma, currently tested in combination with Novartis anti-PD1 mAb spartalizumab
  • TT-10 and TT-53 from Tarus Therapeutics
  • AB928 (Etrumadenant) from Arcus Biosciences, dual A2A/A2B receptor antagonist

Immunotherapy pipeline targeting G Protein-Coupled Receptor Adenosine Receptor A2BR (ADORA2B)

 

Adenosine A2B receptors (A2BRs) gene is located on 17p12-p11.2 locus in human chromosome.

 

The A2B receptor appears to be widely distributed, in the peripheral organs, such as the bowel, bladder, lung, and vas deferens. A2B receptors are found in astrocytes and it seems likely that the cAMP accumulation observed in brain slices is largely due to effects on glial cells. It was shown that the A2B receptor is also a netrin receptor, a family of laminin-related secreted proteins (17).

 

A2BAR is highly expressed in tumor cells, promotes tumor-associated M2 macrophages and tumor growth through VEGF and IL-8.

 

Several antagonists blocking A2BR are currently in clinical trials including:

  • PBF-1129 from Palobiofarma in metastatic lung cancer
  • TT-4 from Tarus Therapeutics for advanced solid tumors
  • AB928 (Etrumadenant) from Arcus Biosciences, dual A2A/A2B receptor antagonist in gastroesophageal and colorectal cancers. 

Nevertheless, due to biased signaling (i.e. new pathways of activation or inhibition), Koussémou et al (18) reported that activated A2BR elicits a reduction in ERK1/2 phosphorylation, an effect that might be exploited in treatment of cancer cell growth and proliferation. Long et al (19) reported on their side that A2BR activation promotes apoptosis mediated by p53.

 

These discoveries pave the way for the development of receptor agonists to A2BAR.

 

Immuno-Oncology Potential of GPCR A3AR (ADORA3): Promising Therapeutic Target?

 

Adenosine A3A receptors (A3ARs) gene is located on 1p21-p13 locus in human chromosome. The Adenosine A3 Receptor (ADORA3) consists of 318 amino acids and shows large interspecies differences in its sequence; the homology between the rat and human A3AR is only 74%.

 

A3ARs are widespread, and the most abundant expression is found in the lung and liver.

The A3AR subtype is widely expressed in a variety of primary cells, tissues, and cell lines. Low levels have been reported in the brain, where it is located in the thalamus, hypothalamus, hippocampus, cortex, and retinal ganglion cells, as well as at motor nerve terminals and the pial and intercerebral arteries. A3ARs are also expressed in microglia and astrocytes, and the inhibition of a neuroinflammatory response in these cells has been associated with their induction of an analgesic effect. At the peripheral level, however, A3AR has been found in enteric neurons, as well as epithelial cells, colonic mucosa, lung parenchyma, and bronchi. Furthermore, A3AR has a broad distribution in inflammatory cells like mast cells, eosinophils, neutrophils, monocytes, macrophages, foam cells, dendritic cells, lymphocytes, splenocytes, bone marrow cells, lymph nodes, synoviocytes, chondrocytes, and osteoblasts, where it mediates anti-inflammatory effects.

 

Interestingly, A3AR is overexpressed in several cancer cells and tissues and is therefore likely to have an important antitumoral role via Wnt/NF-κB pathway modulation (20).

 

The most famous agonists for A3AR are namodenoson and piclidenoson (generically known as IB-MECA and Cl-IB-MECA), currently in clinical trials for different cancers and developed by Can-Fite.

 

Nevertheless, ADORA3 seems to have a dual nature, as the A3AR antagonist approach has also shown some promises (21). Evidence for both pro-tumoral and also anti-tumoral activity suggests other cellular mechanisms are involved in the anticancer properties of these A3AR ligands, and more in-vivo investigation needs to be done. Nevertheless, it should be noted that extrapolation from preclinical animals to humans is hazardous due to the differential expression and function of A3ARs across the animal kingdom.

 


REFERENCES

 

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(2) Pohle. Verh. d. d. Physiol. Ges. 1927; see Ronas Berichte, 42. p. 561.

(3) Bass. Arch. f. Exp. Path. u. Pharm. 76. p. 40. 1914.

(4) Scheele, V. Q. Examen Chemicum Calculi Urinari, Opuscula, 1776, 2, 73

(5) Drury AN, Szent-Györgyi A. The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol. 1929 Nov 25;68(3):213-37. doi: 10.1113/jphysiol.1929.sp002608. PMID: 16994064; PMCID: PMC1402863.

(6) Burnstock, Geoffrey. “A basis for distinguishing two types of purinergic receptor.” (1978).

(7) Libert F, Parmentier M, Lefort A, Dinsart C, Van Sande J, Maenhaut C, Simons MJ. Dumont JE and Vassart G (1989). Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244: 569-572.

(8) Zhou, Q. Y., Li, C., Olah, M. E., Johnson, R. A., Stiles, G. L., & Civelli, O. (1992). Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proceedings of the National Academy of Sciences, 89(16), 7432–7436. doi:10.1073/pnas.89.16.7432

(9) Presta M, Rusnati M, Belleri M, Morbidelli L, Ziche M, and Ribatti D. Purine analogue 6-methylmercaptopurine riboside inhibits early and late phases of the angiogenesis process. Cancer Res 59: 2417–2424, 1999.

(10) Cronstein, Bruce N. "Adenosine receptors and wound healing." TheScientificWorldJOURNAL 4 (2004): 1-8.

(11) Grant MB, Davis MI, Caballero S, Feoktistov I, Biaggioni I, and Belardinelli L. Proliferation, migration, and ERK activation in human retinal endothelial cells through A2B adenosine receptor stimulation. Invest Ophthalmol Vis Sci 42: 2068–2073, 2001.

(12) Symons JD, Firoozmand E, and Longhurst JC. Repeated dipyridamole administration enhances collateral-dependent flow and regional function during exercise. A role for adenosine. Circ Res 73: 503–513, 1993.

(13) Blay J, White TD, Hoskin DW. The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res. 1997;57:2602–5.

(14) Merighi S, Mirandola P, Varani K, Gessi S, Leung E, Baraldi PG, Tabrizi MA, and Borea PA. A glance at adenosine receptors: novel target for antitumor therapy. Pharmacol Ther 100: 31–48, 2003.

(15) Ni S, Wei Q, Yang L. ADORA1 Promotes Hepatocellular Carcinoma Progression via PI3K/AKT Pathway. Onco Targets Ther. 2020;13:12409-12419. Published 2020 Dec 1. doi:10.2147/OTT.S272621

(16) Shi, L.S.; Wu, Z.Y.; Miao, J.; Du, S.C.; Ai, S.C.; Xu, E.; Feng, M.; Song, J.; Guan, W.X. Adenosine interaction with adenosine receptor A2a promotes gastric cancer metastasis by enhancing PI3K-AKT-mTOR signaling. Mol. Biol. Cell 2019, 30, 2527–2534.

(17) Corset, V., Nguyen-Ba-Charvet, K., Forcet, C. et al. Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor. Nature 407, 747–750 (2000). https://doi.org/10.1038/35037600

(18) Koussémou, M., & Klotz, K.-N. (2019). Agonists activate different A2B adenosine receptor signaling pathways in MBA-MD-231 breast cancer cells with distinct potencies. Naunyn-Schmiedeberg’s Archives of Pharmacology. doi:10.1007/s00210-019-01695-2 

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(20) Ren T, Tian T, Feng X, Ye S, Wang H, Wu W, et al. An adenosine A3 receptor agonist inhibits DSS-induced colitis in mice through modulation of the NF-κB signaling pathway. Sci Rep. 2015;5:9047

(21) Merighi S, Benini A, Mirandola P, Gessi S, Varani K, Leung E, Maclennan S, Borea PA. Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells. Biochem Pharmacol. 2006 Jun 28;72(1):19-31. doi: 10.1016/j.bcp.2006.03.020. Epub 2006 Mar 29. PMID: 16682012.